Novel System for the Biocontrol of Pathogens in Aquaculture and Other Animal Systems

ABSTRACT

The inventive technology relates to novel paratransgenic strategies for the biocontrol of pathogens in animal systems using interfering RNA molecules expressed in genetically modified bacteria that may be configured to colonize a target host. In one preferred embodiment, the invention includes novel paratransgenic strategies for the biocontrol of pathogens in aquatic organisms raised in aquaculture environments.

This application claims the benefit of and priority to U.S. ProvisionalApplication No's. 62/480,138, filed Mar. 31, 2017. The entirespecification and figures of the above-referenced application are herebyincorporated, in their entirety by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety.

TECHNICAL FIELD

Generally, the inventive technology relates to novel strategies fordisease control in animal systems. Specifically, the inventivetechnology relates to novel methods, systems and compositions for thebiocontrol of aquatic pathogens in aquatic systems. Specifically, theinvention may comprise novel techniques, systems, and methods for thebiocontrol of disease-transmitting pathogens affecting shrimp inaquaculture systems.

BACKGROUND

The development of aquaculture has generated a significant shift inglobal food production away from traditional catch production methods.Driven primarily by population increases, as well as a lack of growth intraditional capture fishery production, aquaculture has expanded rapidlyto become a major component in the world-wide food productioneco-system. Aquaculture is now seen as playing a key role in manyemerging economies, by virtue of its potential to contribute toincreased food production while helping reduce pressure on fishresources. As noted by the United Nations Food and AgricultureOrganization (UNFAO) in its 2016 Report on the State of the WorldFisheries and Aquaculture, aquaculture is the fastest growing area ofanimal protein production and has significantly outpaced traditionalcapture fishery production. For example, the UNFAO estimates thataquaculture production now accounts for half of all seafood produced forhuman consumption.

The increasing global population, growing demand for seafood, andlimitations on production from capture fisheries will inevitably leadthe continued global expansion of aquaculture with its associated risksof disease emergence and spread. Despite the world's growing reliance onaquaculture as a primary source of food production, especially in manydeveloping economies, traditional aquaculture systems present severaltechnical and biological challenges that limit their overalleffectiveness.

One major drawback of aquaculture systems is that the aquatic animalsare typically placed in high density production systems. This can resultin stress from crowding and sub-optimal water quality conditions thatprovide for easy transmission of disease. In particular, diseaseoutbreaks in aquaculture systems can result in massive losses amongaquatic populations, resulting in large economic losses in commercialaquaculture. Indeed, such disease outbreaks have reportedly cost theaquaculture industry tens of billions dollars in the last 20 years.

In the case of shrimp aquaculture, the problem of disease is especiallysevere. According to the UNFAO, although global aquaculture shrimpproduction has increased, major producing countries, particularly inAsia, have experienced a significant decline in output as a result ofwidespread shrimp disease. There are several reasons for this.

First, unlike vertebrates, shrimp lack many of the key components ofadaptive and innate immune response mechanisms preventing manytraditional methods of inducing or enhancing natural disease resistance.

Second, most of the major pathogenic viruses cause very low levelpersistent infections that can occur at moderate to very high prevalencein apparently healthy shrimp populations. The majority of shrimppathogens are transmitted vertically and disease is the result of amassive viral amplification that follows exposure to various forms ofenvironmental or physiological stress. Stressors can include handling,spawning, poor water quality, or abrupt changes in temperature orsalinity. Shrimp viruses can also be transmitted horizontally. Onceviral loads are high and disease is manifest, horizontal transmission ofinfection is accompanied by transmission of disease.

Third, shrimp commonly are infected simultaneously or sequentially withmultiple viruses, or even different strains of the same virus. This factposes significant challenges for diagnosis, detection, and pathogenexclusion in aquaculture systems.

As one example among many, white spot syndrome (WSS) is a viral diseasecaused by white spot syndrome virus (WSSV). WSSV is a major pathogen inshrimp that causes high mortality and huge economic losses in shrimpaquaculture. The WSS virion is a nonoccluded ellipsoid- orbacilliform-shaped enveloped particle about 275 nm in length and 120 nmin width. Its circular double-stranded DNA consists of 300 kbp coveringapproximately 185 open reading frames (ORFs). WSSV is currently one ofthe most significant impediments to the economical sustainability andgrowth of the global crustacean aquaculture trade. It has caused theloss of billions in USD since its original outbreaks in the early 1990s.To date, WSSV outbreaks have been detected in most major shrimpproducing areas including Asia, Central, South and North America, Europeand Africa.

Traditional efforts to prevent and treat shrimp pathogens, such as WSSV,have been met with limited success. For example, attempts to reduceenvironmental and physiological stressors has been limited due to theeconomic production needs of aquaculture systems as well as a lack oftechnical expertise and appropriate aquaculture facilities in manydeveloping countries. Other attempts have been made to create andisolate pathogen-free populations for aquaculture.

However, such efforts are slow and require significant expertise anddiagnostic capabilities that are prohibitively expensive. Large-scaleapplications of antibiotics have been applied to shrimp aquaculture, inparticular during the production cycle, both in the larval and growthphases. However, the use of antibiotics in aquaculture has beenassociated with environmental and human health problems, includingbacterial resistance, and persistence of the disease in the aquaticenvironment. The accumulation of antibiotic residues in the edibletissues of shrimp may also alter human intestinal flora and cause foodpoisoning or allergy problems. Most importantly, antibiotics areineffective against viruses. Other methods such as the application ofimmunostimulants or bacteriophage treatments to target specificpathogens have been tried with limited commercial and practical success.

An effective system for the biocontrol of pathogens in aquaculture andother animal systems may be: 1) pathogen (virus)-specific so as to notkill off-target organisms; 2) robust or catalytic in mode of action; 3)stable and not easily lost throughout development of the target animal;4) efficient to deliver; 5) simple to manage and low cost; and 6)self-sustainable or regenerating.

To that end, methods of regulating gene expression in pathogens may bean avenue to address the concerns addressed above. Regulating geneexpression either by increasing expression or decreasing expression ofgenes responsible for virulence is considered beneficial for treatmentof diseases. This is especially important in those diseases in whichmaster regulatory genes have been identified. While a majority ofefforts have been extended toward enhancing gene expression,down-regulating specific gene expression is equally important. Anaturally occurring gene-silencing mechanism triggered bydouble-stranded RNA (dsRNA), designated as small interfering RNA(siRNA), has emerged as a very important tool to suppress or knock downgene expression in many systems. RNA interference is triggered by dsRNAthat is cleaved by an RNase-III-like enzyme, Dicer, into 21-25nucleotide fragments with characteristic 5′ and 3′ termini. These siRNAsact as guides for a multiprotein complex, including a PAZ/PIWI domaincontaining the protein Argonaute2, that cleaves the target mRNA. Thesegene-silencing mechanisms are highly specific and potent and canpotentially induce inhibition of gene expression throughout an organism.

The last two decades have also seen tremendous progress in geneexpression technology, including the continued development of bothnon-viral and viral vectors. The non-viral approach to gene expressioninvolves the use of plasmid DNAs (pDNAs), which have a number ofadvantages, including ease of use and preparation, stability and heatresistance, and unlimited size. The plasmids do not replicate inmammalian hosts and do not integrate into host genomes, yet they canpersist in host cells and express the cloned gene for a period of weeksto months.

One area that has seen renewed interest in the use of inhibitory RNAmolecules is infectious diseases, and in particular, pathogens thataffect aquaculture populations. Such strategies for the biocontrol ofpathogens may include paratransgenesis and/or the application ofparatransgenic principles. Paratransgenesis generally refers to systemswhereby symbiotic bacteria, or bacteria capable of colonizing the hostfor a sufficient amount of time to delivery a therapeutic molecule suchas a dsRNA, are genetically modified and reintroduced in thepathogen-bearing host or a pathogen-susceptible population, such asshrimp in aquaculture, where they express effector molecules. However,paratransgenesis has several technical limitations. For example,bacteria to be used in paratransgenesis must generally have three keycomponents: an effector molecule that achieves the desired effect; amechanism to display or excrete the effector molecule; and bacteria thatcan survive in the host long enough to produce the expected amount ofeffector molecules and thereby achieve the desired effect in the host.Therefore, finding such suitable bacteria that fit all of these criteriais very difficult.

Paratransgenesis is generally understood as a technique that attempts toeliminate a pathogen from vector populations through transgenesis of asymbiont of the vector. The goal of this technique is to controlvector-borne diseases. The first step is to identify proteins thatprevent the vector species from transmitting the pathogen. The genescoding for these proteins are then introduced into the symbiont, so thatthey can be expressed in the vector. The final step in the strategy isto introduce these transgenic symbionts into vector populations in thewild. Characteristics of a successful n order to performparatransgenesis may include:

-   -   The symbiotic bacteria can be grown in vitro easily.    -   They can be genetically modified, such as through transformation        with a plasmid containing the desired gene.    -   The engineered symbiont is stable and safe.    -   The association between vector and symbiont cannot be        attenuated.    -   Field delivery is easily handled.

A paratransgenic system is a system that can achieve paratransgenesis ina target organism. Identification of suitable commensal bacteria thatare non-pathogenic to humans or animals among the many organisms that ahost may harbor, particularly in their digestive systems, is paramountfor the success of a paratransgenic system. For example, the chosenbacteria should be capable of colonizing a wide variety of shrimpspecies so that they can be deployed in different species and isolatedstrains.

Furthermore, a well-designed paratransgenic system must also ensure thatthe effector molecule does not interfere with any critical host process,such as reproduction and the like. Such technical and physiologicalchallenges make the development of paratransgenic systems extremelydifficult. Importantly, these technical issues are such that manyparatransgenic systems are neither effective nor appropriate as aneffective biocontrol strategy, especially in complex organisms likeshrimp. These difficulties may also prevent many paratransgenic systemsfrom being appropriately scaled-up to be effective for environmentaldeployment. Generally, biocontrol means utilizing disease-suppressivemicroorganisms to eliminate, control or prevent infection, expressionand/or transmission of selected pathogens.

The foregoing problems regarding the biocontrol of pathogens inaquaculture and other animal systems may represent a long-felt need foran effective—and economical—solution to the same. While implementingelements may have been available, actual attempts to meet this need mayhave been lacking to some degree. This may have been due to a failure ofthose having ordinary skill in the art to fully appreciate or understandthe nature of the problems and challenges involved. As a result of thislack of understanding, attempts to meet these long-felt needs may havefailed to effectively solve one or more of the problems or challengeshere identified. These attempts may even have led away from thetechnical directions taken by the present inventive technology and mayeven result in the achievements of the present inventive technologybeing considered to some degree an unexpected result of the approachtaken by some in the field.

As will be discussed in more detail below, the current inventivetechnology overcomes the limitations of traditional pathogen controlsystems, while meeting the objectives of a truly effective vectorbiocontrol strategy.

SUMMARY OF THE INVENTION(S)

One aim of the present invention may include novel paratransgenicbiocontrol strategies. In this embodiment, the inventive technologyincludes various cross-kingdom mechanisms for the knockdown of essentialviral genes. This may be accomplished through the introduction ofengineered microorganisms into host populations that express specificinhibitory RNA molecules that may downregulate and/or suppress selectedviral and/or host genes.

Another aim of the present invention may include novel paratransgenicbiocontrol strategies for aquaculture populations. In this embodiment,the inventive technology includes various cross-kingdom mechanisms forthe knockdown of essential viral genes in aquatic animals grown inaquaculture systems. This may be accomplished through the introductionof engineered microorganisms into aquaculture animal populations thatexpress specific inhibitory RNA molecules that may downregulate and/orsuppress selected viral and/or host genes.

Another specific aim of the invention may provide a novel paratransgenicsystem that may suppress expression and propagation of the WSSV, amongother viral pathogens, in shrimp aquaculture populations. This systemmay include the introduction of a genetically-modified bacteriatransformed to express select dsRNAs that may target and suppress one ormore pathogen virus genes. Such targets may include, but not be limitedto, the generally conserved region of the WSSV genome coding for: viralcapsid protein 19 (vp19), and/or viral capsid protein 19 gene (vp28)and/or early non-structural gene 477 (Wsv477).

Another aim of the invention may include methods of targeting multipleessential virus-specific gene targets for silencing, such that it may bepossible to selectively diminish viral pathogens in adult shrimppopulations grown in aquaculture environments. In this embodiment, theinvention may include the generation of feeds containing geneticallymodified bacteria configured to express select dsRNAs that may targetand suppress one or more pathogen virus genes. In one embodiment, such atreated feed may be introduced to a pathogen-susceptible orpathogen-affected population. In such an embodiment, feeds containinggenetically modified bacteria configured to express select dsRNAs thatmay target and suppress one or more WSSV genes may be introduced toWSSV-susceptible or WSSV-affected shrimp aquaculture populations. AssiRNAs may be catalytic in activity, their potential effectiveness andsafety may well be greater that traditional viral control methodscurrently employed within the aquaculture industry. In addition, incertain embodiments the inhibitory RNAs are non-immunogenic, such thatthey can be designed to be species specific so that non-target organismsare not harmed and/or affected. Finally, since a bacterial-based dsRNAdelivery system may be self-sustaining and long-lasting, many fewer feedapplications may be needed. In some cases, a single feeding may only beneeded.

In some cases, such interfering RNA molecules, such as dsRNA, may act asa vaccine immunizing individual animals. As such, one aim of theinvention may include the use of genetically modified bacteria tocolonize and express dsRNA molecules that provided individual or herdimmunity in aquatic animals, such as shrimp populations grown inaquaculture systems.

Another aim of the invention may be the generation of geneticallymodified symbiotic and/or probiotic bacterial strain that may expressone or more inhibitory RNA molecules. In a certain embodiment, a shrimpprobiotic bacteria such as Bacillus subtilis, may be geneticallymodified to express one or more inhibitory RNA molecules directed toessential WSSV genes.

Another aim of the invention present inventive technology may includesystems and methods for introducing inhibitory RNA molecules into atarget host through infection by genetically engineered microorganisms.In one embodiment, the invention may provide for genetically engineeredmicroorganisms that may express one or more inhibitory RNA moleculeswithin a target organism. Such target organisms may include aquaticanimals, aquatic animals in aquaculture systems as well as othervertebrate and invertebrate animals generally.

Another aim of the invention may include methods and compositions forthe creation of inhibitory RNA molecules, such as dsRNA, may initiatebiological processes that may inhibit or knock-down essential geneexpression, typically by causing the destruction of specific mRNAmolecules within the cell in multiple animal systems. Additionalembodiments may introduce inhibitory RNA molecules into a targetorganism that inhibit genes necessary for pathogen virulence in multipleanimal systems. Such embodiments may include introduction of inhibitoryRNA molecules into target organisms that may target virulence genes,viral coat proteins, fungal cell wall genes, pathogenic component genes,species-specific metabolic genes and the like.

Additional aims of the invention may include improved delivery systemsfor inhibitory RNA molecules, for example through the use of stabilizingfactors, such as stabilizing proteins and the like. Another preferredembodiment of the inventive technology may include improved systems tofacilitate the transmission of inhibitory RNA molecules within thetarget organism. Yet further embodiments may include geneticallymodified microorganisms that may include genetic constructs that mayfurther co-express certain proteins having processing enzymaticactivity. Such co-expressed proteins may include enzymes that mayinhibit and/or enhance post-translational processing and/or modificationof inhibitory RNA molecules. Other similar embodiments may include theintroduction of microorganisms into a target organism that may express,or even over-express, various genes that may enhance mobilization ofinhibitory RNA molecules and/or genes that may activate secondarydownstream host genes that may target pathogenic pathways.

Finally, the present inventors describe embodiments of the inventionincluding protocol for shrimp feeding by a RNaselll deficient Bacillussubtilis strain (BG322) expressing WSSV-specific dsRNA, and shrimp WSSVchallenge experiments. The present inventors demonstrate, that in thisembodiment, when given with feed including BG322, this geneticallymodified bacteria is able to survive and persist in shrimp intestines.The present inventors further demonstrated that the mortality count fromWSSV challenge experiments for shrimp fed by bacteria expressingWSSV-targeting dsRNA compare to shrimp fed by bacteria expressingunspecific dsRNA indicates a 50% decrease in mortality for shrimp fromfirst group. Additionally, the present inventors show 3-4 log reductionsin viral replication in shrimp fed by WSSV-specific dsRNA. The presentinventors further demonstrate that the presence of WSSV-specificfraction of siRNA in total RNA extracted from shrimp fed by WSSV-dsRNA,which siRNAs are absent in shrimp fed by unspecific dsRNA or shrimp notfed by any dsRNA. Taken together, in these embodiments, the presentinventors demonstrate that delivery of virus-specific dsRNA by entericintestine-colonizing bacteria is an efficient platform for immunizingand/or treating viral shrimp infections.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures, which are incorporated into and form a part ofthe specification, illustrate one or more embodiments of the presentinvention and, together with the description, serve to explain theprinciples of the invention. The drawings are only for the purpose ofillustrating one or more preferred embodiments of the invention and arenot to be construed as limiting the invention. In the figures:

FIG. 1: Total mortality on last day of viral challenge for shrimp fed byBG322 expressing unspecific dsLuc dsRNA or dsRNA specific to WSSV: i)dsVp19 (SEQ ID NO. 3); ii) dsVp28 (SEQ ID NO. 2); and iii) dsWsv477 (SEQID NO. 1);

FIG. 2: PAD-dsRNA plasmid map;

FIG. 3: PAD-43-25 (pGFP expressing) plasmid map;

FIG. 4: Relative WSSV titer in shrimp fed by BG322 expressing unspecificdsLuc dsRNA or dsRNA specific to WSSV (dsVp19, dsVp28 and dsWsv477) atday 10 after WSSV injection. Viral copy numbers were adjusted to log 10scale to allow graphical representation of all groups on the same scale;

FIG. 5: Accumulation of small RNA in BG322 vp19-fed WSSV infectedshrimp. Right panel: RNA electrophoresis on 15% polyacrylamide-8 M ureagel stained by GelRed Nucleic Acid stain (Thermo Fisher Scientific). (L)Takara 14-30 ssRNA Ladder; (1-3) BG322-dsVp19 fed shrimp infected byvirus, (4) Control shrimp (no bacteria, no WSSV), (5) nsRNA (dsLuc) fedshrimp infected by WSSV. Left panel: Northern blotting detection ofvp19-releted siRNA in shrimp: 1-3, (3) BG322-dsVp19 fed shrimp infectedby virus, (4) Control: shrimp (no BG322, no WSSV), (5) Specificitycontrol: BG322-dsLuc fed shrimp infected by WSSV. Arrows indicatevp19-specific bands in siRNA fraction;

FIG. 6: Schematic for selective inactivation of target gene using dsRNAto initiate RNA interference response in a target host cell;

FIG. 7: Schematic for demonstrating typical life-cycle for shrimp.

MODE(S) FOR CARRYING OUT THE INVENTION(S)

The present invention includes a variety of aspects, which may becombined in different ways. The following descriptions are provided tolist elements and describe some of the embodiments of the presentinvention. These elements are listed with initial embodiments, howeverit should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described systems, techniques,and applications. Further, this description should be understood tosupport and encompass descriptions and claims of all the variousembodiments, systems, techniques, methods, devices, and applicationswith any number of the disclosed elements, with each element alone, andalso with any and all various permutations and combinations of allelements in this or any subsequent application.

In one embodiment, the inventive technology may comprise systems,methods and compositions to control specific pathogens by selectiveinactivation of pathogenic, essential or other genes. This targeted geneinactivation may be accomplished by the expression and delivery ofinhibitory RNA molecules, such as double stranded RNA (dsRNA) or smallhairpin RNA (shRNA), to the target host cells where pathogen replicationmay occur. As generally shown in FIG. 6, in a target host, the dsRNA maybe processed into small interfering RNAs (siRNAs) of ˜approximately 21nucleotides in length through the action of the enzyme, Dicer. ThesesiRNAs may further interact with the Ago and RISC protein complexes tobind to the targeted pathogen-specific mRNA sequence. Finally, the RISCcomplex may cleave the pathogen-specific mRNA, silencing or knockingdown the expression of the targeted pathogenic or other target gene andblocking pathogen virulence, replication and/or proliferation.

Additional embodiments may generally include gene inactivation of one,or a plurality of target genes. For example, in one preferredembodiment, gene inactivation may be directed to one or more pathogengenes that are essential to virulence, coat proteins, metabolicactivity, infection pathways and/or energy-production and the like.

Delivery of the inhibitory RNA molecules to a target animal/cell/tissuemay be accomplished through a trans-kingdom delivery system. In apreferred embodiment, the delivery of inhibitory RNA molecules may beaccomplished through the introduction of genetically modifiedhost-specific microorganisms, such as enteric or other bacteria. Sincebacteria cannot process dsRNA to siRNA as they lack the Dicer/RISCmachinery, dsRNA delivered to a target host must be processed by thehost into siRNAs that may inactivate the targeted viral gene. Suchgenetically modified host-specific microorganisms may generally bereferred to as probiotic bacteria or genetically modified bacteria,which may include: 1) microorganisms that are part of the target animalsnormal internal or external bacterial microbiome; 2) microorganisms thathave been modified to be capable of colonizing a target animal, tissue,cell or host environment; 3) microorganisms that that are utilized as afood or energy source by the target host; or 4) microorganisms that havebeen modified to colonize a specific animal, tissue, cell or hostenvironment. In a preferred embodiment, one or more enteric bacteria maybe selected from the group: Acidimicrobiia; Actinobacteria;Alphaprotcobactcria; Anaerolineae; Bacilli; Bactcroidia;Betaproteobacteria; Clostridia; Deltaproteobacteria;Epsilonproteobacteria; Flavobacteria; Fusobactcria; Gammaproteobacteria;Mollicutes; Opitutac; Oscillatoriophycideae; Phycisphaerae;Planctomycetia; Rubrobactcria; Sphingobactcriia; Synechococcophycideae;Thermomicrohia; and Verrucomicrobiae.

The inventive technology may, in a preferred embodiment, include thetrans-kingdom delivery of inhibitory RNA molecules, such as dsRNA,shRNA, siRNA and micro RNAs, for shrimp and other aquatic organisms. Inone embodiment, the invention may comprise a dsRNA-mediated diseasecontrol system that may be configured to inactivate one or more viralgene targets in aquatic organisms such as shrimp. In one preferredembodiment, the invention may comprise a dsRNA-mediated disease controlsystem that may be configured to inactivate one or more viral genetargets in aquatic organisms such as shrimp grown in aquaculture.Examples of shrimp-specific target pathogens may include, but not belimited to: white spot syndrome virus (WSSV); infectious hypodermal andhematopoietic necrosis virus (IHHNV); yellow head virus (YHV); Taurasyndrome virus (TSV); and infectious myonecrosis virus (IMNV) and thelike.

The inventive technology may also comprise a system for thetrans-kingdom delivery of inhibitory RNA molecules to other aquaticvertebrates. In certain embodiments, examples of aquatic vertebratepathogens may include, but not be limited, to those causing viralhemorrhagic septicemia, infectious pancreatic necrosis, viremia of carp,infectious hematopoietic necrosis virus, channel catfish virus, grasscarp hemorrhagic virus, nodaviridae such as nervous necrosis virus orstriped jack nervous necrosis virus, infectious salmon anaemia virus;and the parasites Ceratomyxa shasta, Ichthyophthirius multifillius,Cryptobia salmositica, Lepeophtheirus salmonis, Tetrahymena species,Trichodina species and Epistylus species, spring viraemia of carp (SVC)and Epizootic haematopoietic necrosis (EHNV) and the like.

As noted above, in a preferred embodiment, one or more inhibitory RNAmolecules, in this instance dsRNA, may be delivered to a targethost/population of shrimp through genetically modified enteric bacteriathat may naturally, or be configured to, colonize in the gut of theshrimp. In this embodiment, once colonized in the host, verticaltransmission of the modified bacteria may be passed to the host'sprogeny, thus naturally replicating the pathogenic resistance tosubsequent generations. In certain embodiments, genetically modifiedbacteria expressing one or more inhibitory RNA molecules may colonize ashrimp throughout its lifecycle. For example, as generally shown in FIG.7, genetically modified bacteria expressing one or more inhibitory RNAmolecules may colonize a shrimp while it is: an egg, a nauplius, aprotozoea, a mysis, paost-larval stage or an adult. In this embodiment,the colonized bacteria may express inhibitory RNA molecules, such asdsRNA/shRNAs, that may further be processed by the host's DICER/RISCcomplex allowing pathogen-specific mRNA silencing/inactivation ofessential pathogen genes. Moreover, these colonized enteric bacteria,having permanently and/or temporarily become a part of the host'snatural microbiome, may continuously deliver the dsRNA molecules via theintestine from the earliest larval stages to the adult stage, providingpathogen-specific mRNA silencing/inactivation of essential pathogengenes throughout the host's lifecycle. In addition, as the entericbacteria vector may be an already naturally occurring part of the host'smicrobiome, its presence may not pose any risk to the organism,environment or end-consumers.

Additionally, in certain embodiments the modified bacteria may also behorizontally transmitted to a host population through the distributionof the modified bacteria in treated feed, or feed containing thegenetically modified bacteria, or propagation of the geneticallymodified bacteria into the environment as excreted animal waste. Such afeature may allow for the one-time or at least only periodicadministration of the modified bacteria to the host and/or host'senvironment generating a significant commercial advantage. The inventivetechnology may further comprise methods and techniques to control thelevels and timing of the expression of inhibitory RNA molecules in thetarget organism.

In one preferred embodiment, the expression of one or more inhibitoryRNA molecules may be under the control of a novel gene switch. This geneswitch may be controlled by a switch molecule, which may be awater-soluble and food-grade molecule that can be added to a hostorganism's environment, such as an aquaculture pond or a food supply.The presence of this switch molecule may activate dsRNA production. Inits absence, dsRNA production may not occur, or may only occur atnegligible levels. In a preferred embodiment, aquaculture shrimp, orother target organisms may be infected with one or more viral targetingdsRNA-producing enteric bacteria while the timing and level ofproduction of pathogen gene inactivating dsRNAs may be regulated by thenovel gene switch.

The inventive technology may include methods and techniques for thegeneration of host-specific bacteria, and in particular, host-specificenteric bacteria that may act as an appropriate delivery vector forinhibitory RNA molecules. As an exemplary model, shrimp may be utilizedas a target host. However, as can be appreciated by one of ordinaryskill in the art, such methods and techniques may be applied to avariety of different organisms.

In this preferred embodiment, one or more shrimp gut samples fromvarious larvae and adult shrimp species may be captured from the wildand characterized to identify the associated bacterial metagenome. Fromthis initial genomic characterization, a list of target bacteria thatinhabit the larval gut, as well as their species-specific relativeabundance may be established. Additional steps in this characterizationmay include comparing and contrasting the metagenome of the entericbacteria from adults metamorphosed from the aforementioned larvae todetermine which bacterial species persist from larvae to adult stagesand in what proportions.

In certain embodiments, a host-specific bacteria may include one or moreof the following characteristics: 1) a dominant bacteria in the gutflora of both the larval and adult stages of shrimp; 2) culturableoutside of the shrimp, for example in a fermenter; 3) no known adverseenvironmental or health impacts on non-target organisms; 4) capable ofbeing genetically engineered to stably express and deliver dsRNA insufficient quantities to inhibit target gene replication, in at leastone, but preferably, all life stages of the shrimp.

Bacterial RNAse Ills may degrade inhibitory RNA molecules such as dsRNA.As such, in one embodiment, the inventive technology may includemodification of the previously identified host-specific bacteria, orprobiotic, to have decreased RNase III expression, or inactivated RNaseIII function or activity. This decrease or inactivation in RNAase IIIexpression and/or activity may inhibit or decrease RNase III-mediatedprocessing of dsRNA into smaller RNA species. In one preferredembodiment, the previously identified host-specific bacteria may begenetically modified to efficiently express inhibitory RNA molecules inan RNAse III deficient background. In this preferred embodiment, theRNAse IIIs genes of the host-specific bacteria may be knocked out byhomologous recombination or other appropriate methods.

Another embodiment of the inventive technology may include systems andmethods to facilitate the overexpression of host-specific bacterialgenes known to enhance stabilization and/or mobilization of inhibitoryRNA molecules. In this preferred embodiment, one or more genes known tostabilize dsRNA may be overexpressed to enhance its lifetime andfacilitate its movement within host organism/cell/tissue. In anotherpreferred embodiment, one or more genes that regulate or suppress genesthat are known to stabilize dsRNA may be knocked-out resulting in theirupregulation thereby enhancing dsRNA's lifetime to facilitate itsmovement within host organisms to enhance the viral gene inhibition.Additional embodiments may also overexpress genes or target geneknockouts that may result in the upregulation of membrane vesiculartrafficking to facilitate dsRNA mobilization and delivery to the hostorganism.

Each of the aforementioned systems may be embodied in genetic constructsthat may include transcription regulation elements such as promoters,terminators, co-activators and co-repressors and other control elementsas may be regulated in prokaryotic as well as eukaryotic systems. Suchsystems may allow for control of the type, timing and amount of,inhibitory RNA molecules or other proteins, expressed within the system.Additional embodiments may include genetic constructs that may beinduced through additional outside and/or environmental factors, such asthe presence of a specific protein or compound, such as stress relatedproteins generated in response to a pathogen or even proteins and otherprecursor compounds generated by pathogens and the like.

Additional optimization procedures of the current inventive technologymay include introducing modified microorganisms into shrimp larvae in alaboratory environment using competition studies with multiple foodtargets and different densities of microorganisms expressing dsRNA, todetermine the effective dosage for suppressing viral replication. Shrimplarval tissues may be analyzed by in situ RNA hybridization to thedetermine the cellular location where gene expression was most impactedby dsRNA to better select additional target genes for dsRNA-mediatedgene suppression.

As noted above, in one embodiment, the present invention includes anovel paratransgenic system which may further include a novel method forimplementation of an RNAi-based strategy in which natural shrimpsymbiotic bacteria are transformed with plasmids that express dsRNA,targeting essential genes that may reduce or eliminate transmission ofsuch pathogens. Such embodiments may have particular application toaquatic organisms in aquaculture environments.

As noted above, aquatic organisms, such as shrimp, possess a naturalanti-viral defense mechanism RNA interference (RNAi). Briefly, by use ofthe exo-siRNA RNAi pathway, shrimp recognize viral long double-stranded(ds)RNA generated during virus replication, digest it to 21-bp shortinterfering RNA (siRNA) segments with an RNase III family enzyme calledDicer 2, and use these as effectors to identify, cleave and inactivatereplicating virus genomes.

Thus, according to one aspect of the present invention, there isprovided a method of controlling a pathogenically infected shrimp, themethod comprising administering to a shrimp population a geneticallymodified bacteria expressing a heterologous nucleic acid sequence whichspecifically downregulates an expression of at least one essentialpathogen gene product of the shrimp, wherein downregulation of theexpression of the at least one essential pathogen resistance gene mayprevent replication and/or pathogenicity of the shrimp pathogen.

In one embodiment, the present invention includes the generation of anovel paratransgenic system for the biocontrol of pathogen-vectors. Theinvention may specifically include a paratransgenic system configured todeliver one or more inhibitory RNA molecules topathogen/disease-transmitting organisms. In one embodiment, theinvention may include one or more genetically engineered microorganismsconfigured to deliver one or more inhibitory RNA molecules to aquaticorganisms in aquaculture systems. In a preferred embodiment, theinvention may include one or more genetically engineered entericbacteria configured to deliver one or more dsRNA molecules to aquaticorganisms in aquaculture systems.

Other embodiments of the current invention include the generation of oneor more enteric bacteria that may be endosymbiotic, or act as aprobiotic as herein defined, with the target host organism, in this caseshrimp. These enteric bacteria may persist in the gut throughout allstages of shrimp development. Another embodiment of the inventionincludes the generation of one or more enteric bacteria that may beendosymbiotic, or act as a probiotic as herein defined, that are furthergenetically modified, or transformed, to produce one or more dsRNA(double-stranded) molecules. These dsRNA molecules may correspond to oneor more pathogen genes. Moreover, dsRNA molecules may generate anRNA-mediated downregulation or suppression of select viral genes. ThisRNA-mediated downregulation or suppression of select viral genes may bethrough an interfering RNA process as generally described here.

Another embodiment includes the generation of one or more entericbacteria that may be endosymbiotic, or act as a probiotic as hereindescribed, with the target host organism and that may colonize the gutof the target host, and further be configured to continuously deliverdsRNA molecules that correspond to one or more pathogen genes of thewhite spot syndrome virus (WSSV), or other aquatic pathogens identifiedherein, and may further elicit an interfering RNA-mediated reactioncausing the suppression of the target viral genes.

In one preferred embodiment, the invention may include methods andcompositions for the biocontrol of WSSV infection in shrimp. In thispreferred embodiment, a shrimp population may be administered agenetically modified bacteria expressing a heterologous nucleic acidsequence which specifically downregulates an expression of at least oneessential WSSV gene product, wherein downregulation of the expression ofthe essential WSSV gene may prevent replication and/or pathogenicity ofWSSV in shrimp.

In this preferred embodiment, the heterologous nucleic acid sequenceexpresses an RNA duplex, comprising a sense region and an antisenseregion, wherein the antisense region includes a plurality of contiguousnucleotides that are complementary to a messenger RNA sequence encodedby the target gene. In one embodiment, the polynucleotide encoding thesiRNA comprises at least one nucleotide sequence configured to generatea hpRNA that targets one or more essential WSSV genes. In this preferredembodiment, such hpRNAs may inhibit expression of target genes in WSSVincluding, but not limited to: viral capsid protein 19 (vp19), viralcapsid protein 19 (vp28), and early non-structural gene (Wsv477) amongothers. In this embodiment, a heterologous nucleic acid sequenceexpresses an RNA duplex, or hpRNA, may be selected from the groupconsisting of: SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3. In thisembodiment, SEQ ID NO.1 is configured to target and inhibit expressionof an early non-structural gene (Wsv477)(SEQ ID. NO 9 and 10). SEQ IDNO.1 is configured to target and inhibit expression of viral capsidprotein 19 (vp28) (SEQ ID. NO 7 and 7), and SEQ ID NO.3 is configured totarget and inhibit expression of viral capsid protein 19 (vp19) (SEQ ID.NO 5 and 6). It should be noted that the identification of a DNAsequence also includes the corresponding RNA sequence it encodes. Assuch, a reference to a SEQ ID NO. that includes DNA also specificallyinclude the sequence of the RNA that it expresses as would be understoodby one of ordinary skill in the art. For example, where it is claimsthat a heterologous inhibitory polynucleotide may be selected from thegroup consisting of: SEQ ID NO. 1, SEQ ID NO. 2, and/or SEQ ID NO. 3,such a claim may include the sequence of the inhibitory RNA molecule asone of ordinary skill could easily determine without undueexperimentation.

In a preferred embodiment, a messenger RNA sequence encoded by thetarget pathogen gene may include a gene located in a region of higherthan average homology, or in other words, a gene fully or partiallylocated in the most conserved region of a pathogens genome, whencompared to the sequences of other strains of the pathogens of genes. Inone specific embodiment, SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO.3,correspond to highly conserved structural and/or non-structural proteinscoding regions in SEQ ID NO. 5-10, generally. Naturally, such sequencesare exemplary, as they may be alternatively, redundant or overlappedacross one or more distinct gene coding segments.

The present invention may further include one or more vectors formodulating multiple pathogen genes, wherein the vector comprising one,or a plurality of dsRNAs may correspond to one or more select pathogengenes, for example the WSSV genes identified in SEQ ID NO. 6, 8, 10. Asgenerally shown in FIGS. 2-3, this embodiment may include the use of aplasmid expression system. In some embodiments, this plasmid may haveone or more expression cassettes, including: at least one genesuppressing cassette containing a polynucleotide operably-linked to apromoter sequence, wherein the polynucleotide encodes an interfering RNAmolecule, such as a dsRNA, or a molecule that will subsequently generateinterfering RNA molecule, such as a dsRNA, that reduces expression of atarget pathogen gene by RNA interference.

A preferred embodiment of the present invention includes a vector formodulating multiple host genes, wherein the vector comprising one, or aplurality of dsRNAs may correspond to one or more select host genes.This embodiment may include the use of a plasmid expression system. Insome embodiments, this plasmid may have one or more expressioncassettes, including: at least one gene suppressing cassette containinga polynucleotide operably-linked to a promoter sequence, wherein thepolynucleotide encodes an interfering RNA molecule, such as a dsRNA, ora molecule that will subsequently generate interfering RNA molecule,such as a dsRNA, that reduces expression of a target host gene by RNAinterference.

Another embodiment of the present invention includes a vector formodulating host and pathogen genes, wherein the vector comprising one,or plurality of dsRNAs that may correspond to one or more select hostand pathogen genes. This embodiment may include the use of a plasmidexpression system. In some embodiments, this plasmid may have one ormore expression cassettes, including: at least one gene suppressingcassette containing a first polynucleotide operably-linked to a promotersequence, wherein the polynucleotide encodes an interfering RNAmolecule, such as a dsRNA, or a molecule that will subsequently generatean interfering RNA molecule, such as a dsRNA, that reduces expression ofa target host gene by RNA interference. This gene cassette may furthercontain a second polynucleotide operably-linked to a promoter sequence,wherein the polynucleotide encodes an interfering RNA molecule, such asa dsRNA, or a molecule that will subsequently generate interfering RNAmolecule, such as a dsRNA, that reduces expression of a target pathogengene by RNA interference.

The present invention also includes a vector for inhibiting theexpression of viral or bacterial genes in a host, wherein the vectorcomprises at least one gene suppressing cassette containing apolynucleotide operably-linked to a promoter sequence, wherein thepolynucleotide encodes an siRNA molecule that reduces expression of atarget pathogen gene within the host by RNA interference. In oneembodiment, the polynucleotide encoding the siRNA comprises thenucleotide sequence of SEQ ID NO.2, SEQ ID NO.2, and/or SEQ ID NO.3.

Likewise, the vectors of the present invention can include a pluralityof gene suppressing cassettes, wherein each gene suppressing cassettecontains a polynucleotide encoding an siRNA molecule, such as a dsRNA,that targets the same mRNA sequence or different mRNA sequences. Forexample, each gene suppressing cassette can encode a dsRNA molecule thattargets an mRNA sequence of two or more different genes. Furthermore,each vector of the present invention can include a plurality of genepromoting cassettes and a plurality of gene suppressing cassettes.

Examples of suitable promoters for gene suppressing cassettes include,but are not limited to, T7 promoter, bla promotor, U6 promoter, pol IIpromoter, Ell promoter, and CMV promoter and the like. Optionally, eachof the promoter sequences of the gene promoting cassettes and the genesuppressing cassettes can be inducible and/or tissue-specific.

An additional aspect of the invention may include novel methods toprovide genetically engineered enteric-bacteria that may be configuredto colonize an animal gut and prevent viral pathogens from escaping thegut into the surrounding epithelium. More specifically, one aim of theinvention may be to introduce genetically engineered enteric-bacteria toa shrimp's gut and be further configured to produce and secrete dsRNA.These dsRNA molecules may be taken up by the surrounding epithelialcells, causing a strong RNAi cascade preventing viral replication,and/or suppressing pathogen levels such that no significant number ofvirions can migrate from the epithelial cells surrounding the shrimp'sgut. In additional embodiments, target epithelial cells may uptake dsRNAsecreted by transformed paratransgenic bacteria located in the gutthrough endocytic, vesicular trafficking, phagocytosis and/or otheractive or passive polynucleotide transport processes.

The present invention, in some embodiments thereof, relates to isolatednucleic acid agents, and, more particularly, but not exclusively, to theuse of the same for controlling pathogenically infected animals, such asshrimp.

According to some embodiments of the invention, an essential gene mayinclude a gene selected from the group consisting of one or more targetpathogen genes that are essential to virulence, coat proteins, metabolicactivity, infection pathways and/or energy-production and the like. Atarget gene may include one or more genes that are responsible forpathogenicity, or the capacity to cause a disease condition in the host.Examples of such target genes may also include one or more virulencefactors. Examples of such virulence factors may include, but not belimited to:

-   -   Adherence Factors: This group may include genes that help        bacterial pathogens adhere to certain cells;    -   Invasion Factors: This group may include genes for surface        components that allow the bacterium to invade host cells;    -   Capsules: This group may include genes for structural capsules        that may protect bacteria from opsonization and phagocytosis;    -   Endotoxins: This group may include genes for several types of        toxic lipopolysaccharides that may elicit an immune response;    -   Exotoxins: This group may include genes for several types of        protein toxins and enzymes produced and/or secreted from        pathogenic bacteria. Major categories include cytotoxins,        neurotoxins, and enterotoxins;    -   Siderophores: This group may include genes for several types of        iron-binding factors that allow some bacteria to compete with        the host for iron, which is bound to hemoglobin, transferrin,        and lactoferrin;    -   Host-Conversion Factors: This group may include genes that alter        the metabolism of the host to the benefit of the pathogen,        including but not limited to evading host defenses.    -   Structural: This group of genes may include genes encoding viral        capsis needed for viral replication.    -   Non-Structural: This group of genes may include genes encoding        non-structural viral proteins needed for viral replication.        Example may include genes responsible for viral genome        integration and/or replication.

One preferred embodiment of the present invention may include anisolated nucleic acid agent, comprising a polynucleotide expressing anucleic acid sequence which specifically downregulates an expression ofat least one pathogen gene. In a preferred embodiment, this isolatednucleic acid agent may comprise a polynucleotide expressing a dsRNAsequence which specifically downregulates an expression of at least oneanimal pathogen through a siRNA process. Another embodiment of thepresent invention may include a nucleic acid construct comprising anucleic acid sequence encoding the isolated nucleic acid agent, such asa dsRNA or a nucleic acid agent that may form into a dsRNA, in someembodiments of the invention.

According to alternative embodiments of the invention, the nucleic acidsequence directly corresponds with a pathogen gene, while in alternativeembodiments the nucleic acid sequence corresponds, or overlaps with oneor more pathogen genes. According to some embodiments of the invention,the dsRNA nucleic acid sequence directly corresponds with a pathogengene, while in alternative embodiments the dsRNA nucleic acid sequencecorresponds, or overlaps with one or more pathogen genes.

As noted above, the inventive technology may be applied to a variety oforganisms, both plant and animal. For example, the invention maycomprise dsRNA-mediated disease control systems that may be configuredto inactivate one or more pathogen gene targets in any appropriate hostorganism. In one specific embodiment, one or more inhibitory RNAmolecules, in this instance dsRNA, may be delivered to a targethost/population of vertebrate and invertebrate animals. For example,poultry such as: chicken, turkey, duck and geese (see Table 4), bees(see FIG. 5) as well as mammals (see FIG. 6), including humans, throughgenetically modified enteric bacteria that may naturally, or beconfigured to colonize in the gut of the host.

In this exemplary embodiment, the colonized bacteria may expressinhibitory RNA molecules, such as dsRNA/shRNAs, that may further beprocessed by the host's DICER/RISC complex allowing pathogen-specificmRNA silencing/inactivation of essential pathogen genes. Examples ofsome poultry specific pathogens that may be targeted by the inventionare listed in tables 4-6 below. These colonized enteric bacteria, havingbecome a part of the host's natural microbiome, may continuously deliverthe dsRNA molecules via the intestine during various stages of thedevelopment providing pathogen-specific mRNA silencing/inactivation ofessential pathogen genes throughout the host's lifecycle. In addition,as the enteric bacteria vector may be an already naturally occurringpart of the poultry's or bee's microbiome, its presence will not poseany risk to the organism, environment or end-consumers, nor will allowfor vertical transmission to progeny and horizontal transmission to hostpopulation at large through the distribution of the modified bacteriaexcreted into the environment as waste.

In certain embodiments, a probiotic bacteria may be configured toexpress one or more interfering RNA molecules. In this preferredembodiment, a subject, such as a human, may take a therapeutic oreffective amount of the probiotic which may act as a vaccine orantibiotic or anti-viral pharmaceutical. Production, and delivery ofsuch probiotic bacteria is well known and would be within the skill onthose generally skilled in the art.

The term “aquaculture” as used herein includes the cultivation ofaquatic organisms under controlled conditions.

The term “aquatic organism” and/or “aquatic animal” as used hereininclude organisms grown in water, either fresh or saltwater. Aquaticorganisms/animals includes vertebrates, invertebrates, arthropods, fish,mollusks, including, shrimp (e.g., penaeid shrimp, Penaeus esculentu,Penaeus setiferus, Penaeus stylirostris, Penaeus occidentalis, Penaeusjaponicus, Penaeus vannamei, Penaeus monodon, Penaeus chinensis, Penaeusaztecus, Penaeus duorarum, Penaeus indicus, and Penaeus merguiensis,Penaeus californiensis, Penaeus semisulcatus, Penaeus monodon, brineshrimp, freshwater shrimp, etc), crabs, oysters, scallop, prawn clams,cartilaginous fish (e.g., sea bream, trout, bass, striped bass, tilapia,catfish, salmonids, carp, catfish, yellowtail, carp zebrafish, red drum,etc), crustaceans, among others. Shrimp include, shrimp raised inaquaculture as well.

The term “probiotic” refers to a microorganism, such as bacteria, thatmay colonize a host for a sufficient length of time to delver atherapeutic or effective amount of an interfering RNA molecule. Aprobiotic may include endosymbiotic bacteria, or naturally occurringflora that may permanently to temporarily colonize an animal, such as anaquatic organism. Probiotic organisms may also include algae, and fungi,such as yeast.

Specific examples of bacterial vectors include bacteria (e.g., cocci androds), filamentous algae and detritus. Specific embodiments oftransformable bacterial vectors cells that may be endogenous through alllife cycles of the host may include all those listed herein. Additionalembodiments may include one or more bacterial strains selected from theexamples listed herein. Naturally, such a list is not exclusive, and ismerely exemplary of certain preferred embodiments of paratransgenicbacterial strains.

As used herein, the phrase “host” refers to an animal carrying adisease-causing pathogen, an animal susceptible to a disease-causingpathogen, an animal population where members are carrying adisease-causing pathogen, or an animal population where members aresusceptible to a disease-causing pathogen. These include hosts listed intables 4-6, and elsewhere.

As used herein, “pathogen” refers to a disease causing agent. Theseinclude the pathogens included in tables 4-6, and elsewhere.

As used herein, “vaccine” refers to compositions that result in bothactive and passive immunizations. Both polynucleotides and theirexpressed gene products are referred to as vaccines herein. A feedincluding a treated bacteria configured to express an interferingbacteria may also be a vaccine. Feeding treated feed to an animal may bea vaccination.

As used herein, the phrase “feed” refers to animal consumable materialintroduced as part of the feeding regimen or applied directly to thewater in the case of aquatic animals. A “treated feed” refers to a feedtreated with a treated bacteria configured to express an interferingbacteria.

As used herein, the term “controlling” and/or “bio-control” refers toreducing and/or regulating pathogen/disease progression and/ortransmission.

The term “nucleic acid” as used herein, refers to a polymer ofribonucleotides or deoxyribonucleotides. Typically, “nucleic acid or“nucleic acid agent” polymers occur in either single or double-strandedform, but are also known to form structures comprising three or morestrands. The term “nucleic acid” includes naturally occurring nucleicacid polymers as well as nucleic acids comprising known nucleotideanalogs or modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, which have similarbinding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Exemplaryanalogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). “DNA”,“RNA”, “polynucleotides”, “polynucleotide sequence”, “oligonucleotide”,“nucleotide”, “nucleic acid”, “nucleic acid molecule”, “nucleic acidsequence”, “nucleic acid fragment”, and “isolated nucleic acid fragment”are used interchangeably herein.

The terms “isolated”, “purified”, or “biologically pure” as used herein,refer to material that is substantially or essentially free fromcomponents that normally accompany the material in its native state orwhen the material is produced. In an exemplary embodiment, purity andhomogeneity are determined using analytical chemistry techniques such aspolyacrylamide gel electrophoresis or high performance liquidchromatography. A nucleic acid or particular bacteria that are thepredominant species present in a preparation is substantially purified.In an exemplary embodiment, the term “purified” denotes that a nucleicacid or protein gives rise to essentially one band in an electrophoreticgel. Typically, isolated nucleic acids or proteins have a level ofpurity expressed as a range. The lower end of the range of purity forthe component is about 60%, about 70% or about 80% and the upper end ofthe range of purity is about 70%, about 80%, about 90% or more thanabout 90%.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, organism,nucleic acid, protein or vector, has been modified by the introductionof a heterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells may express genes thatare not found within the native (non-recombinant or wild-type) form ofthe cell or express native genes that are otherwise abnormallyexpressed, over-expressed, under expressed or not expressed at all.

The terms “genetically modified,” “bio-transformed,” “transgenic”,“transformed”, “transformation”, and “transfection” are similar inmeaning to “recombinant”. “Transformation”, “transgenic”, and“transfection” refer to the transfer of a polynucleotide into the genomeof a host organism or into a cell. Such a transfer of polynucleotidescan result in genetically stable inheritance of the polynucleotides orin the polynucleotides remaining extra-chromosomally (not integratedinto the chromosome of the cell). Genetically stable inheritance maypotentially require the transgenic organism or cell to be subjected fora period of time to one or more conditions which require thetranscription of some or all of the transferred polynucleotide in orderfor the transgenic organism or cell to live and/or grow. Polynucleotidesthat are transformed into a cell but are not integrated into the host'schromosome remain as an expression vector within the cell. One may needto grow the cell under certain growth or environmental conditions inorder for the expression vector to remain in the cell or the cell'sprogeny. Further, for expression to occur the organism or cell may needto be kept under certain conditions. Host organisms or cells containingthe recombinant polynucleotide can be referred to as “transgenic” or“transformed” organisms or cells or simply as “transformants”, as wellas recombinant organisms or cells.

A genetically altered organism is any organism with any change to itsgenetic material, whether in the nucleus or cytoplasm (organelle). Assuch, a genetically altered organism can be a recombinant or transformedorganism. A genetically altered organism can also be an organism thatwas subjected to one or more mutagens or the progeny of an organism thatwas subjected to one or more mutagens and has changes in its DNA causedby the one or more mutagens, as compared to the wild-type organism (i.e,organism not subjected to the mutagens). Also, an organism that has beenbred to incorporate a mutation into its genetic material is agenetically altered organism.

The term “vector” refers to some means by which DNA, RNA, a protein, orpolypeptide can be introduced into a host. The polynucleotides, protein,and polypeptide which are to be introduced into a host can betherapeutic or prophylactic in nature; can encode or be an antigen; canbe regulatory in nature; etc. There are various types of vectorsincluding virus, plasmid, bacteriophages, cosmids, and bacteria.

An “expression vector” is a nucleic acid capable of replicating in aselected host cell or organism. An expression vector can replicate as anautonomous structure, or alternatively can integrate, in whole or inpart, into the host cell chromosomes or the nucleic acids of anorganelle, or it is used as a shuttle for delivering foreign DNA tocells, and thus replicate along with the host cell genome. Thus,expression vectors are polynucleotides capable of replicating in aselected host cell, organelle, or organism, e.g., a plasmid, virus,artificial chromosome, nucleic acid fragment, and for which certaingenes on the expression vector (including genes of interest) aretranscribed and translated into a polypeptide or protein within thecell, organelle or organism; or any suitable construct known in the art,which comprises an “expression cassette”. In contrast, as described inthe examples herein, a “cassette” is a polynucleotide containing asection of an expression vector of this invention. The use of thecassette assists in the assembly of the expression vectors. Anexpression vector is a replicon, such as plasmid, phage, virus, chimericvirus, or cosmid, and which contains the desired polynucleotide sequenceoperably linked to the expression control sequence(s).

A polynucleotide sequence is “operably linked to an expression controlsequence(s)” (e.g., a promoter and, optionally, an enhancer) when theexpression control sequence controls and regulates the transcriptionand/or translation of that polynucleotide sequence. As used herein, thephrase “gene product” refers to an RNA molecule or a protein.

This invention utilizes routine techniques in the field of molecularbiology. Basic texts disclosing the general methods of use in thisinvention include Green and Sambrook, 4th ed. 2012, Cold Spring HarborLaboratory; Kriegler, Gene Transfer and Expression: A Laboratory Manual(1993); and Ausubel et al., eds., Current Protocols in MolecularBiology, 1994-current, John Wiley & Sons. Unless otherwise noted,technical terms are used according to conventional usage. Definitions ofcommon terms in molecular biology maybe found in e.g., Benjamin Lewin,Genes IX, published by Oxford University Press, 2007 (ISBN 0763740632);Krebs, et al. (eds.), The Encyclopedia of Molecular Biology, publishedby Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

The present teachings contemplate the targeting of homologs andorthologs according to the selected host species. Homologous sequencesinclude both orthologous and paralogous sequences. The term “paralogous”relates to gene-duplications within the genome of a species leading toparalogous genes. The term “orthologous” relates to homologous genes indifferent organisms due to ancestral relationship. Thus, orthologs areevolutionary counterparts derived from a single ancestral gene in thelast common ancestor of given two species (Koonin EV and Galperin MY(Sequence—Evolution—Function: Computational Approaches in ComparativeGenomics. Boston: Kluwer Academic; 2003. Chapter 2, Evolutionary Conceptin Genetics and Genomics. Available from:ncbi(dot)nlm(dot)nih(dot)gov/books/NBK20255) and therefore have greatlikelihood of having the same function. As such, orthologs usually playa similar role to that in the original species in another species.

Homology (e.g., percent homology, sequence identity+sequence similarity)can be determined using any homology comparison software computing apairwise sequence alignment. As used herein, “sequence identity” or“identity” in the context of two nucleic acid or polypeptide sequencesincludes reference to the residues in the two sequences which are thesame when aligned. When percentage of sequence identity is used inreference to proteins it is recognized that residue positions which arenot identical often differ by conservative amino acid substitutions,where amino acid residues are substituted for other amino acid residueswith similar chemical properties (e.g. charge or hydrophobicity) andtherefore do not change the functional properties of the molecule. Wheresequences differ in conservative substitutions, the percent sequenceidentity may be adjusted upwards to correct for the conservative natureof the substitution. Sequences which differ by such conservativesubstitutions are to have “sequence similarity” or “similarity”. Meansfor making this adjustment are well-known to those of skill in the art.Typically this involves scoring a conservative substitution as a partialrather than a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., according tothe algorithm of Henikoff S and Henikoff JG. [Amino acid substitutionmatrices from protein blocks. Proc. Natl. Acad. Sci. U.S.A. 1992,89(22): 10915-9],

According to a specific embodiment, the homolog sequences are at least60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or even identical to thesequences (nucleic acid or amino acid sequences) provided herein.Homolog sequences of SEQ ID Nos 1-6 of between 50%-99% may be includedin certain embodiments of the present invention.

Downregulating expression of a pathogen resistance gene product of ahost can be monitored, for example, by direct detection of genetranscripts (for example, by PCR), by detection of polypeptide(s)encoded by the gene (for example, by Western blot orimmunoprecipitation), by detection of biological activity ofpolypeptides encode by the gene (for example, catalytic activity, ligandbinding, and the like), or by monitoring changes in the hosts (forexample, reduced mortality of the host etc). Additionally oralternatively downregulating expression of a pathogen resistance geneproduct may be monitored by measuring pathogen levels (e.g. virallevels, bacterial levels etc.) in the host as compared to wild type(i.e. control) hosts not treated by the agents of the invention.

As generally noted above, according to some aspects of the invention,there is provided an isolated nucleic acid agent comprising a nucleicacid sequence, which specifically downregulates the expression of atleast one host pathogen resistance gene product. According to oneembodiment, the agent is a polynucleotide agent, such as an RNAsilencing agent. In a preferred embodiment, the agent is apolynucleotide agent, such as dsRNA, configured to induce RNAinterference.

As used herein, the term “interfering RNA molecules” or “interferingRNA” refers to an RNA which is capable of inhibiting or “silencing” theexpression of a target gene. In certain embodiments, the RNA silencingagent is capable of preventing complete processing (e.g. the fulltranslation and/or expression) of an mRNA molecule through apost-transcriptional silencing mechanism. RNA silencing agents includenoncoding RNA molecules, for example RNA duplexes comprising pairedstrands, as well as precursor RNAs from which such small noncoding RNAscan be generated. Exemplary RNA silencing agents include dsRNAs such assiRNAs, miRNAs and shRNAs. In one embodiment, the RNA silencing agent iscapable of inducing RNA interference. In another embodiment, the RNAsilencing agent is capable of mediating translational repression.

In some embodiments of the invention, the nucleic acid agent is a doublestranded RNA (dsRNA). As used herein the term “dsRNA” relates to twostrands of anti-parallel polyribonucleic acids held together by basepairing. Examples include SEQ ID NOs 1-5. The two strands can be ofidentical length or of different lengths provided there is enoughsequence homology between the two strands that a double strandedstructure is formed with at least 60%, 70% 80%, 90%, 95% or 100%complementary over the entire length. According to an embodiment of theinvention, there are no overhangs for the dsRNA molecule. According toanother embodiment of the invention, the dsRNA molecule comprisesoverhangs. According to other embodiments, the strands are aligned suchthat there are at least 1, 2, or 3 bases at the end of the strands whichdo not align (i.e., for which no complementary bases occur in theopposing strand) such that an overhang of 1, 2 or 3 residues occurs atone or both ends of the duplex when strands are annealed.

It will be noted that the dsRNA can be defined in terms of the nucleicacid sequence of the DNA encoding the target gene transcript, and it isunderstood that a dsRNA sequence corresponding to the coding sequence ofa gene comprises an RNA complement of the gene's coding sequence, orother sequence of the gene which is transcribed into RNA.

The inhibitory RNA sequence can be greater than 90% identical, or even100% identical, to the portion of the target gene transcript.Alternatively, the duplex region of the RNA may be defined functionallyas a nucleotide sequence that is capable of hybridizing with a portionof the target gene transcript under stringent conditions (e.g., 400 mMNaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 60 degrees C. hybridization for12-lb hours; followed by washing). The length of the double-strandednucleotide sequences complementary to the target gene transcript may beat least about 18, 19, 21, 25, 50, 100, 200, 300, 400, 491, 500, 550,600, 650, 700, 750, 800, 900, 1000 or more bases. In some embodiments ofthe invention, the length of the double-stranded nucleotide sequence isapproximately from about 18 to about 530, or longer, nucleotides inlength.

The present teachings relate to various lengths of dsRNA, whereby theshorter version i.e., x is shorter or equals 50 bp (e.g., 17-50), isreferred to as siRNA or miRNA. Longer dsRNA molecules of 51-600 arereferred to herein as dsRNA, which can be further processed for siRNAmolecules. According to some embodiments, the nucleic acid sequence ofthe dsRNA is greater than 15 base pairs in length. According to yetother embodiments, the nucleic acid sequence of the dsRNA is 19-25 basepairs in length, 30-100 base pairs in length, 100-250 base pairs inlength or 100-500 base pairs in length. According to still otherembodiments, the dsRNA is 500-800 base pairs in length, 700-800 basepairs in length, 300-600 base pairs in length, 350-500 base pairs inlength or 400-450 base pairs in length. In some embodiments, the dsRNAis 400 base pairs in length. In some embodiments, the dsRNA is 750 basepairs in length.

The term “siRNA” refers to small inhibitory RNA duplexes (generallybetween 17-30 base pairs, but also longer e.g., 31-50 bp) that inducethe RNA interference (RNAi) pathway. Typically, siRNAs are chemicallysynthesized as 21mers with a central 19 bp duplex region and symmetric2-base 3′-overhangs on the termini, although it has been recentlydescribed that chemically synthesized RNA duplexes of 25-30 base lengthcan have as much as a 100-fold increase in potency compared with 21mersat the same location. The observed increased potency obtained usinglonger RNAs in triggering RNAi is theorized to result from providingDicer with a substrate (27mer) instead of a product (21mer) and thatthis improves the rate or efficiency of entry of the siRNA duplex intoRISC. It has been found that position of the 3′-overhang influencespotency of a siRNA and asymmetric duplexes having a 3′-overhang on theantisense strand are generally more potent than those with the3′-overhang on the sense strand (Rose et al., 2005). This can beattributed to asymmetrical strand loading into RISC, as the oppositeefficacy patterns are observed when targeting the antisense transcript.

In certain embodiment, dsRNA can come from 2 sources; one derived fromgene transcripts generated from opposing gene promoters on oppositestrands of the DNA and 2) from fold back hairpin structures producedfrom a single gene promoter but having internal complimentary. Forexample, strands of a double-stranded interfering RNA (e.g., a siRNA)may be connected to form a hairpin or stem-loop structure (e.g., ashRNA). Thus, as mentioned the RNA silencing agent may also be a shorthairpin RNA (shRNA). The term “shRNA”, as used herein, refers to an RNAagent having a stem-loop structure, comprising a first and second regionof complementary sequence, the degree of complementarity and orientationof the regions being sufficient such that base pairing occurs betweenthe regions, the first and second regions being joined by a loop region,the loop resulting from a lack of base pairing between nucleotides (ornucleotide analogs) within the loop region. The number of nucleotides inthe loop is a number between and including 3 to 23, or 5 to 15, or 7 to13, or 4 to 9, or 9 to 11. Some of the nucleotides in the loop can beinvolved in base-pair interactions with other nucleotides in the loop.Examples of oligonucleotide sequences that can be used to form the loopinclude 5′-UUCAAGAGA-3′ (Brummelkamp, T. R. et al. (2002) Science 296:550, SEQ ID NO. 302) and 5′-UUUGUGUAG-3′ (Castanotto, D. et al. (2002)RNA 8:1454, SEQ ID NO. 303). It will be recognized by one of skill inthe art that the resulting single chain oligonucleotide forms astem-loop or hairpin structure comprising a double-stranded regioncapable of interacting with the RNAi machinery.

As used herein, the phrase “microRNA” (also referred to hereininterchangeably as “miRNA” or “miR”) or a precursor thereof” refers to amicroRNA (miRNA) molecule acting as a post-transcriptional regulator.Typically, the miRNA molecules are RNA molecules of about 20 to 22nucleotides in length which can be loaded into a RISC complex and whichdirect the cleavage of another RNA molecule, wherein the other RNAmolecule comprises a nucleotide sequence, essentially complementary tothe nucleotide sequence of the miRNA molecule. Typically, a miRNAmolecule is processed from a “pre-miRNA” or as used herein a precursorof a pre-miRNA molecule by proteins, such as DCL proteins, and loadedonto a RISC complex where it can guide the cleavage of the target RNAmolecules. Pre-microRNA molecules are typically processed frompri-microRNA molecules (primary transcripts). The single stranded RNAsegments flanking the pre-microRNA are important for processing of thepri-miRNA into the pre-miRNA. The cleavage site appears to be determinedby the distance from the stem-ssRNA junction (Han et al. 2006, Cell 125,887-901, 887-901).

As used herein, a “pre-miRNA” molecule is an RNA molecule of about 100to about 200 nucleotides, preferably about 100 to about 130 nucleotides,which can adopt a secondary structure comprising an imperfect doublestranded RNA stem and a single stranded RNA loop (also referred to as“hairpin”), and further comprising the nucleotide sequence of the miRNA(and its complement sequence) in the double stranded RNA stem. Accordingto a specific embodiment, the miRNA and its complement are located about10 to about 20 nucleotides from the free ends of the miRNA doublestranded RNA stem. The length and sequence of the single stranded loopregion are not critical and may vary considerably, e.g. between 30 and50 nucleotides in length. The complementarity between the miRNA and itscomplement need not be perfect, and about 1 to 3 bulges of unpairednucleotides can be tolerated. The secondary structure adopted by an RNAmolecule can be predicted by computer algorithms conventional in the artsuch as mFOLD. The particular strand of the double stranded RNA stemfrom the pre-miRNA which is released by DCL activity and loaded onto theRISC complex is determined by the degree of complementarity at the 5′end, whereby the strand, which at its 5′ end, is the least involved inhydrogen bonding between the nucleotides of the different strands of thecleaved dsRNA stem, is loaded onto the RISC complex and will determinethe sequence specificity of the target RNA molecule degradation.However, if empirically the miRNA molecule from a particular syntheticpre-miRNA molecule is not functional (because the “wrong” strand isloaded on the RISC complex), it will be immediately evident that thisproblem can be solved by exchanging the position of the miRNA moleculeand its complement on the respective strands of the dsRNA stem of thepre-miRNA molecule. As is known in the art, binding between A and Uinvolving two hydrogen bonds, or G and U involving two hydrogen bonds isless strong that between G and C involving three hydrogen bonds.

Naturally occurring miRNA molecules may be comprised within theirnaturally occurring pre-miRNA molecules, but they can also be introducedinto existing pre-miRNA molecule scaffolds by exchanging the nucleotidesequence of the miRNA molecule normally processed from such existingpre-miRNA molecule for the nucleotide sequence of another miRNA ofinterest. The scaffold of the pre-miRNA can also be completelysynthetic. Likewise, synthetic miRNA molecules may be comprised within,and processed from, existing pre-miRNA molecule scaffolds or syntheticpre-miRNA scaffolds. Some pre-miRNA scaffolds may be preferred overothers for their efficiency to be correctly processed into the designedmicroRNAs, particularly when expressed as a chimeric gene wherein otherDNA regions, such as untranslated leader sequences or transcriptiontermination and polyadenylation regions are incorporated in the primarytranscript in addition to the pre-microRNA.

According to the present teachings, the dsRNA molecules may be naturallyoccurring or synthetic. The dsRNA can be a mixture of long and shortdsRNA molecules such as, dsRNA, siRNA, siRNA+dsRNA, siRNA+miRNA, or acombination of same.

In a preferred embodiment, one or more nucleic acid agents are designedfor specifically targeting a target gene of interest (e.g. a pathogennon-structural gene). It will be appreciated that the nucleic acid agentcan be used to downregulate one or more target genes (e.g. as describedin detail above). If a number of target genes are targeted, aheterogenic composition which comprises a plurality of nucleic acidagents for targeting a number of target genes is used. Alternatively theplurality of nucleic acid agents is separately formulated. According toa specific embodiment, a number of distinct nucleic acid agent moleculesfor a single target are used, which may be used separately orsimultaneously (i.e., co-formulation) applied.

For example, in order to silence the expression of an mRNA of interest,synthesis of the dsRNA suitable for use with some embodiments of theinvention can be selected as follows. First, the mRNA sequence isscanned including the 3′ UTR and the 5′ UTR. Second, the mRNA sequenceis compared to an appropriate genomic database using any sequencealignment software, such as the BLAST software available from the NCBI.Putative regions in the mRNA sequence which exhibit significant homologyto other coding sequences are filtered out. Qualifying target sequencesare selected as templates for dsRNA synthesis. Preferred sequences arethose that have as little homology to other genes in the genome as toreduce an “off-target” effect.

It will be appreciated that the RNA silencing agent of some embodimentsof the invention need not be limited to those molecules containing onlyRNA, but further encompasses chemically-modified nucleotides andnon-nucleotides.

According to one embodiment, the dsRNA specifically targets a geneselected from the group consisting of SEQ ID Nos 1-4 or a variant ofhomolog thereof. In addition, the term AMPLICON means a piece of DNA orRNA.

In certain embodiments, expression of the dsRNA molecule doesn't requirea cis-acting regulatory sequence (e.g., heterologous) transcribing thedsRNA. As used herein, the term “heterologous” refers to exogenous,not-naturally occurring within a native cell of the host or in a cell inwhich the dsRNA is fed to a host (such as by position of integration, orbeing non-naturally found within the cell).

The nucleic acid agent can be further comprised within a nucleic acidconstruct comprising additional regulatory elements. For example,transcription from an expression cassette, a regulatory region (e.g.,promoter, enhancer, silencer, leader, intron and polyadenylation) may beused to modulate the transcription of the RNA strand (or strands).Therefore, in one embodiment, there is provided a nucleic acid constructcomprising the nucleic acid agent. The nucleic acid construct can havepolynucleotide sequences constructed to facilitate transcription of theRNA molecules of the present invention operably linked to one or morepromoter sequences functional in a host cell. The polynucleotidesequences may be placed under the control of an endogenous promoternormally present in the host genome. The polynucleotide sequences of thepresent invention, under the control of an operably linked promotersequence, may further be flanked by additional sequences thatadvantageously effect its transcription and/or the stability of aresulting transcript. Such sequences are generally located upstream ofthe promoter and/or downstream of the 3′ end of the expressionconstruct. The term “operably linked,” as used in reference to aregulatory sequence and a structural nucleotide sequence, means that theregulatory sequence causes regulated expression of the linked structuralnucleotide sequence.

Genetic “control elements” refer to nucleotide sequences locatedupstream, within, or downstream of a structural nucleotide sequence, andwhich influence the timing and level or amount of transcription, RNAprocessing or stability, or translation of the associated structuralnucleotide sequence. Regulatory sequences may include promoters,translation leader sequences, introns, enhancers, stem-loop structures,repressor binding sequences, termination sequences, pausing sequences,polyadenylation recognition sequences, and the like.

It will be appreciated that the nucleic acid agents can be delivered tothe animal, such as a shrimp in a variety of ways. According to oneembodiment, the composition of some embodiments comprises cells, whichcomprise the nucleic acid agent. As used herein, the term “cell” or“cells,” with respect to a host may refer to an animal cell in any stageof its lifecycle. In a certain embodiment, the paratransgenic system mayestablish genetically modified bacteria that may be endogenous throughall life cycles of the host. According to a specific embodiment, thecell is a bacterial cell.

In a further embodiment, a composition including a genetically modifiedbacteria configured to express dsRNA may be formulated as feed and/or awater dispersible granule or powder that may further be configured to bedispersed into the environment. In yet a further embodiment, thecompositions of the present invention may also comprise a wettablepowder, spray, emulsion, colloid, aqueous or organic solution, dust,pellet, or colloidal concentrate. Dry forms of the compositions may beformulated to dissolve immediately upon wetting, or alternatively,dissolve in a controlled-release, sustained-release, or othertime-dependent manner.

Alternatively or additionally, the composition may comprise an aqueoussolution. Such aqueous solutions or suspensions may be provided as aconcentrated stock solution which is diluted prior to application, oralternatively, as a diluted solution ready-to-apply. Such compositionsmay be formulated in a variety of ways. They may be employed as wettablepowders, granules or dusts, by mixing with various inert materials, suchas inorganic minerals (silicone or silicon derivatives, phyllosilicates,carbonates, sulfates, phosphates, and the like) or botanical materials(powdered corncobs, rice hulls, walnut shells, and the like). Theformulations or compositions containing paratransgenic bacteria mayinclude spreader-sticker adjuvants, stabilizing agents, other pesticidaladditives, or surfactants. Liquid formulations may be employed as foams,suspensions, emulsifiable concentrates, or the like. The ingredients mayinclude Theological agents, surfactants, emulsifiers, dispersants, orpolymers.

According to one embodiment, the composition is administered to the hostby feeding. Feeding the host with the composition can be effected once,regularly, or semi-regularly over the span of hours, days, weeks, monthsor even years.

As mentioned, the dsRNA of the invention may be administered as a nakeddsRNA. Alternatively, the dsRNA of the invention may be conjugated to acarrier known to one of skill in the art, such as a transfection agente.g. PEI or chitosan or a protein/lipid carrier or coupled tonanoparticles. The compositions may be formulated prior toadministration in an appropriate means such as lyophilized,freeze-dried, microencapsulated, desiccated, or in an aqueous carrier,medium or suitable diluent, such as saline or other buffer. Suitableagricultural carriers can be solid, semi-solid or liquid and are wellknown in the art. Such compositions may be considered“agriculturally-acceptable carriers”, which may covers all adjuvants,e.g., inert components, dispersants, surfactants, tackifiers, binders,etc. that are ordinarily used in pesticide formulation technology.

According to some embodiments, the nucleic acid agent is provided inamounts effective to reduce or suppress expression of at least one hostpathogen resistance gene product. As used herein “a suppressive amount”or “an effective amount” or a “therapeutically effective amount” refersto an amount of dsRNA which is sufficient to downregulate (reduceexpression of) the target gene by at least 20%, 30%, 40%, 50%, or more,say 60%, 70%, 80%, 90% or more even 100%, or reduce mortality in ananimal or animal population, such as shrimp in aquaculture by at least ameasurable percentage, preferably between 1%-100%.

Testing the efficacy of gene silencing can be affected using any methodknown in the art. For example, using quantitative RT-PCR measuring geneknockdown. Thus, for example, host animals from each treatment group canbe collected and pooled together. RNA can be extracted therefrom andcDNA syntheses can be performed. The cDNA can then be used to assess theextent of RNAi, by measuring levels of gene expression using qRT-PCR.Reagents of the present invention can be packed in a kit including thenucleic acid agent (e.g. dsRNA), instructions for administration of thenucleic acid agent, construct or composition to a specific host.

As used herein, the term “gene” or “polynucleotide” refers to a singlenucleotide or a polymer of nucleic acid residues of any length. Thepolynucleotide may contain deoxyribonucleotides, ribonucleotides, and/ortheir analogs, and may be double-stranded or single stranded. Apolynucleotide can comprise modified nucleic acids (e.g., methylated),nucleic acid analogs or non-naturally occurring nucleic acids and can beinterrupted by non-nucleic acid residues. For example, a polynucleotideincludes a gene, a gene fragment, cDNA, isolated DNA, mRNA, tRNA, rRNA,isolated RNA of any sequence, recombinant polynucleotides, primers,probes, plasmids, and vectors. Included within the definition, arenucleic acid polymers that have been modified, whether naturally or byintervention.

Constructs of the invention may be prepared by any method known in theart for the synthesis of DNA and RNA molecules, including techniques forchemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art, for example, solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors which incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines. Moreover, various well-knownmodifications to nucleic acid molecules may be introduced as a means ofincreasing intracellular stability and half-life. Possible modificationsinclude, but are not limited to, the addition of flanking sequences ofribonucleotides or deoxyribonucleotides to the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the oligodeoxyribonucleotide backbone.

As used herein, the terms “approximately” or “about” refer to ±10%.Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals there between.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”. The term“consisting of” means “including and limited to”. The term “consistingessentially of” means that the composition, method or structure mayinclude additional ingredients, steps and/or parts, but only if theadditional ingredients, steps and/or parts do not materially alter thebasic and novel characteristics of the claimed composition, method orstructure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences, unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity, andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range, such as from 1 to 6 should be considered to havespecifically disclosed subranges such as from 1 to 3, from 1 to 4, from1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well asindividual numbers within that range, for example, 1, 2, 3, 4, 5, and 6.This applies regardless of the breadth of the range.

As used herein, the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts. As used herein, the term “treating”includes abrogating, substantially inhibiting, slowing or reversing theprogression of a condition, substantially ameliorating clinical oraesthetical symptoms of a condition or substantially preventing theappearance of clinical or aesthetical symptoms of a condition.

The invention now being generally described will be more readilyunderstood by reference to the following examples, which are includedmerely for the purposes of illustration of certain aspects of theembodiments of the present invention. The examples are not intended tolimit the invention, as one of skill in the art would recognize from theabove teachings and the following examples that other techniques andmethods can satisfy the claims and can be employed without departingfrom the scope of the claimed invention. Indeed, while this inventionhas been particularly shown and described with references to preferredembodiments thereof, it will be understood by those skilled in the artthat various changes in form and details may be made therein withoutdeparting from the scope of the invention encompassed by the appendedclaims.

EXAMPLES Example 1: Generation of Expression Vectors and GeneticallyModified Bacterial Strain

Plasmids for dsRNA expression were made by cloning dsRNA sequencebetween 2 converging pUpp promoters in pAD-43-25 plasmid. As generallyshown in FIGS. 2-3, PAD-dsRNA plasmid map and PAD-43-25 (pGFPexpressing) plasmid map were generated by the present inventors. RNaseIII deficient Bacillus subtilis strain BG322 was transformed withpAD-dsRNA plasmids (3 plasmids for WSSV-specific dsRNAexpression—pAD-dsWsv477 (SEQ ID NO. 1), pAD-dsVp28 (SEQ ID NO. 2),pAD-dsVp19 (SEQ ID NO. 3), and plasmid for expression of unspecificdsLuc pAD-dsLuc (SEQ ID NO. 4).

Example 2: Preparation of Shrimp Feed Containing Genetically ModifiedBacteria

Bacteria were grown overnight in LB with 5 mg/ml chloramphenicol, andthen centrifuged and mixed into a common commercial shrimp feed (ZeiglerPL 40) at a concentration of 1E+08 CFU/gm feed and refrigerated.Prepared feed was fed to one gram shrimp at 10% body weight, dividedinto three feedings per day.

Example 3: Colonization and Persistence of Genetically Modified Bacteriain Shrimp Intestine

The present inventors have demonstrated that the exemplary geneticallymodified B. subtilis strain BG322 is able to colonize and persist inshrimp intestines. In this embodiment, the present inventors transformedBG322 with plasmid pAD-43-25 encoding the fluorescent GFP protein.Shrimp were provided feed containing BG322 pAD-43-25 for 10 days andpresence of BG322 was detected at days 5 and 10 by analysis of shrimpintestines under fluorescent microscope (GFP fluorescence detected inthe intestines) and by plate count method (BG322 colonies wereidentified by GFP fluorescence) using isolated guts of shrimp. After 5days of BG322 feeding the bacterial titer in the BG322 intestines was˜1.1E+06 cfu/g and it stayed at approximately the same tier on day 10(Table 1). Since the bacteria concentration in shrimp intestines becameconstant after 5 days of feeding, day 5 after feeding bacteria waschosen by the present inventors as an appropriate day for beginning theviral challenges.

Example 4: Introduction of dsRNA Expression Vectors to Shrimp

SPF shrimp (Shrimp Improvement System, Islamorada, Fla.) 1 g weight weremaintained in 7 gallon aquariums (n=12). Shrimp were randomly assignedto five treatments groups: 1) shrimp fed with commercial food, no virusinjections; 2) shrimp fed with commercial food with BG322pAD-dsLuc/virus injections; 2) shrimp fed commercial feed with BG322pAD-dsVp19/virus injections; 3) shrimp fed by commercial food with BG322pAD-dsVp28/virus injections; 4) shrimp fed by commercial food with BG322pAD-dsWsv477/virus injections and 5) shrimp fed with commercial foodwith BG322 pAD-dsLuc/virus injections. Bacteria were provided to shrimpfor 5 days before viral injection and during the course of the challengevia feed. Such shrimp being generally referred to as “treated shrimp.”

Example 5: WSSV Challenge in dsRNA Expressing (Treated) Shrimp

The present inventors exposed treated shrimp to WSSV by injection ofinoculant derived from infected tissues (Ecuador 2001 strain) of shrimpand stored at −80° C. The tissues were homogenized in a blender in a1:10 ratio of tissue to UV treated chilled salt well water for 30 sec.The preparation was poured into a 50 ml tube and centrifuged for 10minute at 2500 RPM at 4° C. The resulting supernatant was filtered witha 0.45 μm (PES) filter (Whatman Puradisc®). The filtrate was injectedintramuscularly between the second and third tail segment at 20 μl/gbody weight of shrimp immediately after preparation.

Example 6: WSSV-Induced Mortality Count in dsRNA Expressing (Treated)Shrimp

Mortality count was performed by the present inventors at day 7-10 afterthe viral injection. In this embodiment, 6 biological replicates(separate tanks) were analyzed for negative control group (no virus), 4replicates for shrimp fed by dsLuc (non-specific dsRNA), and 5replicates for each shrimp group fed by BG322-dsVp19, BG322-dsVp28 orBG322-dsWsv477 (anti-WSSV specific dsRNA).

Mortality count from last days of time-course shows ˜50% reduction inmortality in shrimp fed by WSSV-targeting dsRNA compared to shrimp fedby unspecific dsRNA. The present inventors further demonstrated 30%, 33%or 38% average mortality was observed in shrimp fed by dsVp19, dsVp28and dsWsv477 correspondently, while dsLuc fed group had 64% mortality.This difference is statistically significant for p<0.05 (FIG. 1). Assuch, the present inventors demonstrate that BG322 expressingvirus-specific dsRNA provide shrimp protection from the virus.

Example 7: Sequence-Specific Inhibition of WSSV Replication in ShrimpFed by BG322 Expressing WSSV-Targeting DNA

The present inventors used qPCR to evaluate the level of virusreplication in shrimp after 10 days of WSSV challenge. Survivors fromgroups fed BG322-dsLuc, BG322-dsVp19, BG322-dsVp28 and BG322-dsWsv477were used for total DNA extraction (n=4-6). Total DNA was extracted withInvitrogen Tissue DNA kit, and the concentration of the extracted DNAswas quantified using a NanoDrop ND-100 spectrophotometer (NanoDropTechnologies). 50-100 ng of total DNA was used as a template for qPCR.SybrGreen assay was carried out with SybrGreen PowerUp master Mix.Primers WSSV1011(5′) and WSSV1079 (3′) generating 69 bp replicon wereused for the reaction (Table 2) (Durand et all, 2002). Cyclingconditions were as follows: 1 initial step at 95° C. for 10 min followedby 40 cycles of 95° C. for 30 s, 58° C. for 30 s and 72° C. for 30 s.Melting temperature of each sample was examined to verify the purity ofthe PCR products. PCR reactions were performed on Stratagene Mx3005real-time PCR system and analyzed with Mx3005 software. For comparison,resulting numbers were normalized to readings from shrimp fed byBG322-dsLUC (positive control group).

The present inventors observed a log 3-4 fold reduction in the amount ofvirus detected in all shrimp groups fed bacteria expressing WSSV-gene(VP19, VP28, wsv477) targeting dsRNA compared to the amount of virus inshrimp fed bacteria expressing unspecific dsLuc dsRNA (negativecontrol). The difference between unspecific dsRNA group and other groupsis statistically significant at p<0.01 as demonstrated generally in FIG.4.

Example 8: Detection of Antiviral Vp19 siRNA in WSSV-Infected Shrimp

The present inventors demonstrated the accumulation of bacteria-specificsmallRNA through Northern blotting analysis of smallRNA samples purifiedfrom BGG322 vp19-fed shrimps that survived infection by WSSV.

In this embodiment, the present inventors prepared DIG 3′ end-labeledprobes directed to small RNAs. Sequences of DNA oligonucleotidescomplimentary to the vp19 dsRNA are presented in Table 3. The probeswere ordered via IDTDNA and labeled with the DIG oligonucleotide 3′-endlabeling kit. Reaction was stopped by addition of 200 mM EDTA (pH 8.0).Labeled DNA probes were stored at −20° C.

Total RNAs were isolated from the shrimp muscle samples by use ofE.Z.N.A miRNA isolation kit (Omega) according to the manufacturer'sinstructions. The quality and integrity of total RNAs were evaluated byelectrophoresis on 15% polyacrylamide-8 M urea gels. The concentrationof the extracted RNAs was quantified using a NanoDrop ND-100spectrophotometer (NanoDrop Technologies). At least 10 μg of total RNAwere electrophoresed in a denaturing 15% polyacrylamide gel containing 8M urea and transferred to a positively charged nylon membrane (Roche).After cross-linking with UV, the membrane was prehybridized inPerfectHyb (Sigma) for 1 h and then hybridized with a digoxigenin(DIG)-labeled DNA probes complementary to dsRNA sequence for 20 h at 44°C. The membranes were washed four subsequent times with SSC wash bufferssupplemented with 0.1% SDS (2×, 0.5×, and 0.1×, respectively). Signaldetection was performed following the instructions for a DIG High PrimeDNA labeling and detection starter kit II (Roche). ssRNA markers of 14nt, 18 nt, 22 nt, 26 nt and 30 nt were used as markers of small RNA(Takara). Finally, the hybridization signals were visualized by BIO-RADChemiDoc XRS.

As generally demonstrated in FIG. 5, the present inventors showedintensive virus specific smallRNAs in the samples that were preparedfrom infected shrimp, confirming that viral infection inhibited vp19gene expression via small interfering RNAs. No prominent small viral RNAwas detected in native shrimp that were not infected with WSSV (lane 4).Accumulation of vp19-specific siRNA with sizes ranging from 26 nt to 18nt was detected in two out of three shrimps (Lanes 1-2). This resultdemonstrated that that BGG322 vp19 antiviral protection is mediated byan siRNA pathway.

Example 9: Enterobacter Ag1 Bacteria are Able to Survive and StablyColonize Shrimp Intestines

In order to check if Ag1 bacteria are able to colonize shrimpintestines, the present inventors performed a colonization experiment.Shrimp were fed by Ag1 labeled by fluorescent EGFP protein for 5 days,afterward bacterial feeding was terminated and shrimp were fed bycommercial food. As shown in Table 5 below, concentration of Ag1 inshrimp intestines was measured by plating and cfu counting for 7 weekspost-feeding. Ag1 was present in shrimp intestines during wholeexperimental time with no noticeable decrease in concentration. Thepresent inventor have concluded that Ag1 bacteria are able tosuccessfully colonize shrimp intestines and therefore are suitablebacterial strain for long-term delivery of therapeutic molecules forshrimp protection.

REFERENCES

The following references are hereby incorporated in their entirety byreference:

-   [1] Sanjuktha M, Stalin Raj V, Aravindan K, Alavandi S V, Poornima    M, Santiago T C. Comparative efficacy of double-stranded RNAs    targeting WSSV structural and nonstructural genes in controlling    viral multiplication in Penaeus monodon. Arch Virol. 2012 May;    157(5):993-8. doi: 10.1007/s00705-012-1258-2. Epub 2012 Feb. 21.    PubMed PMID: 22350694.-   [2] Kumar, Anil & Allnutt, F C Thomas & Sayre, Richard. (2015).    Double Stranded RNA Simultaneously Targeting Four White Spot    Syndrome Virus (WSSV) genes provides Protection against WSSV in    Litopenaeus vannamei. International Journal of Marine Science and    Ocean Technology. 2. 5-10.-   [3] Lezzerini M, van de Ven K, Veerman M, Brul S, Budovskaya YV.    Specific RNA Interference in Caenorhabditis elegans by Ingested    dsRNA Expressed in Bacillus subtilis. PLoS One. 2015 Apr. 30;    10(4):e0124508. doi: 10.1371/journal.pone.0124508. eCollection 2015.    PubMed PMID: 25928543; PubMed Central PMCID: PMC4416053.-   [4] Durand, S. V. and Lightner, D. V. (2002), Quantitative real time    PCR for the measurement of white spot syndrome virus in shrimp.    Journal of Fish Diseases, 25: 381-389.    doi:10.1046/j.1365-2761.2002.00367.x.-   [5] Chakraborty A., Otta S. K., Kumar B. J. S., Hossain M. S.,    Karunasagar I., Venugopal M. N., Karunasagar I., Prevalence of white    spot syndrome virus in wild crustaceans along the coast of India,    Curr. Sci. (2002) 82:1392-1397.).-   [6] Nguyen D V, Christiaens O, Bossier P and Smagghe G (2016) RNA    interference in shrimp and potential applications in aquaculture.    Reviews in Aquaculture (2016) 0: 1-12. doi: 10.1111/raq.12187.-   [7] Sanchez-Martinez J. G., Aguirre-Guzman G., Mejia-Ruiz H., White    spot syndrome virus in cultured shrimp: a review, Aquac. Res. (2007)    38:1339-1354.-   [8] Flegel T. W., Nielsen L., Thamavit V., Kongtim S., Pasharawipas    T., Presence of multiple viruses in non-diseased, cultivated shrimp    at harvest, Aquaculture (2004) 240:55-68.-   [9] Hoa T., Hodgson R., Oanh D., Phuong N., Preston N., Walker P.,    Genotypic variations in tandem repeat DNA segments between    ribonucleotide reductase subunit genes of white spot syndrome virus    (WSSV) isolates from Vietnam, in: Walker P., Lester R., Reantaso M.    (Eds.), Diseases in Asian Aquaculture, Asian Fisheries Society,    Manila, 2005, pp. 395-403.-   [10] Ma, D.; Hu, Y.; Wang, J.; Ye, S. & Li, A. (2006). Effects of    antibacterials use in aquaculture on biogeochemical processes in    marine sediment. The Science of the Total Environment, vol. 367, No.    1, pp. 273-277.-   [11] Martinez Cruz, Patricia et al. “Use of Probiotics in    Aquaculture.” ISRN Microbiology 2012 (2012).-   [12] Kobayashi, M. & Brummett, R. 2014. Disease management in    aquaculture. In: Forum for Agricultural Risk Management in    Development.-   [13] Walker, Peter J., and James R. Winton. “Emerging Viral Diseases    of Fish and Shrimp.” Veterinary Research 41.6 (2010).

Tables

TABLE 1 Fluorescence load in intestines of shrimp fed by GFP-expressingBG322 Fluorescent load, Fluorescent load, Group 5 days, cfu/g 10 days,cfu/g shrimp fed by BG322-GFP 1.1E+06 2.1E+05 shrimp fed by commercialfeed 0 0

TABLE 2 Primers used in qPCR reaction WSSV1011 TGGTCCCGTCCTCATCTCAGWSSV1079 GCTGCCTTGCCGGAAATTA

TABLE 3 Oligonucleotides used in micro RNANorthern blot analysis and complimentary to dsRNA vp19 Name DNA sequenceVP19-1 GCCACCACGACTAACACTCTTCCTTTCGGC AGGACCGGAGCCCAGGCCGCTGGCCCTTCTVP19-2 AGACCCATGCGAGCCATAGACATGGAGCCT TCAAGATCTTCCATGGTGTAAGAAGGGCCAVP19-3 GCATGGGTCTCTTTTTGATCGTTGCTATCT CAATTGGTATCCTCGTCCTGGCCGTCATGAVP19-4 GTCCTTATCAGTGTCAGAATCGCTGTCCTT CTTTGGTCCCATCCATACATTCATGACGGCVP19-5 TGATAAGGACACCGATGATGATGACGACAC TGCCAACGATAACGATGATGAGGACAAATAVP19-6 ACAGAAGAGCGGACCCAGCCAGAAGCATCA TATCCCTGGTCCTGTTCTTATATTTGTCCTVP19-7 GGCTGGGTCCGCTCTTCTGTTCCTCGTTTC CGCCGCCACCGTTTTTATGTCTTACCCCAA

TABLE 4 Target pathogens in poultry populations Poultry Viral diseasesFungal diseases Parasitic diseases Chickens Avian influenzaAspergillosis Coccidiosis (genus: Turkeys (has multiple strains (genus:Aspergillus) Eimeria) Ducks or types, and is divided into three types:A, B, and C; H5N1 (genus: Influenzavirus A) can cause a 90- 100%mortality) Newcastle Disease Candidiasis (genus: Ascaridia galli (genus:Avulavirus) Candida) (genus: Ascaridia) Poxvirus diseases (mainlygenera: Parapoxvirus, Blackhead (genus: orthopoxvirus, Histomonas)yatapoxvirus, molluscipoxvirus) Infectious Mites (genus: bronchitisvirus Dermanyssus) (IBV) (genus: Gammacoronavirus) LaryngotracheitisLice (genus: (genus: Iltovirus) Menophon) Marek's Disease (genus:Mardivirus) Eastern Equine Encephalitis (genus: Alphavirus) Hemorrhagicenteritis (genus: Siadenovirus) Viral arthritis (genus: Reovirus)

TABLE 5 Target pathogens in bee populations Bees (Apis Mellifera) Viraldiseases Fungal diseases Parasitic diseases Dicistroviruses: Nosemaapis- causing nosemosis, Varroa mite the most common (Varroadesfructor). adult honey bees disease. Israeli acute Ascosphaera apisHoney bee tracheal paralysis virus (causing mites (Acarapis (CCD (ColonyChalkbrood disease) woodi) Collapse Syndrom)) Kashmir bee virusAspegillus spp Small hive beetles (CCD) (causing (Aethina tumida)Stonebrood disease) colonies damage in non-apis bees (bumble bees andstingless bees) Acute bee Tropilaelaps mites paralysis virus(Tropilaelaps (CCD) mercedesae) Black queen cell Wax moth virus (affectpupae (Pyralidae: Galleria but not adults) Mellonela and Achroiagrisella) Aphid lethal paralysis virus (possibly CCD) Big sioux rivervirus (possibly CCD) Iflaviruses: Deformed wing virus Kakugo virusVarroa destructor virus-1 Sacbrood virus Thai/Chinese sacbrood virusSlow bee paralysis virus Baculovirus: Apis iridescent virus (CCD)Unclassified viruses: Cloudy wing virus Bee virus-X Bee virus-Y LakeSinai virus-1 Lake Sinai virus-2

TABLE 6 Target pathogens in mammal populations Mammal DiseasesBluetongue Virus (BTV): Affects sheep, goats, deer and cattle BovineViral Diarrhoea (BVD): Cattle and other ruminants Calf Pneumonia: Causedby bovine Respiratory Syncytial Virus (bRSV), Parainfluenza III Virus(PI3) Infectious Bovine Rhinotracheitis (IBR): Caused by BovineHerpesvirus-1 (BHV-1) Trypanosomosis (Sleeping disease): Affects bothhuman and animals Transmitted through tse-tse fly by flagellatedprotozoan parasites. The most economically important livestock diseaseof Africa Foot-and-mouth disease Virus (FMDV): Highly contagious viraldisease that affects cattle and swine. It also affects sheep, goats,deer, and other cloven-hooved ruminants Rift Valley Fever Virus: viraldisease of cattle and sheep. It is spread through infected mosquitoes.It can spread to humans either as airborne and/or by consuming raw milk,handling undercooked meat. Rotaviral Diarrhoea: Caused by bovineRotavirus Parasitic gastro-enteritis (PGE or Gut worms): Affect cattleand is spread through parasites (abomasal worms) Anaplasmosis:Vector-borne, infectious blood disease in cattle caused by therickettsial parasites Anaplasma marginale and Anaplasma centrale. It isalso known as yellow-bag or yellow-fever Bovine Anaemia: Benigntheileriosis is a tick-borne disease caused by intracellular bloodparasites belonging to the Theileria orientalis group (BATOG) BovineBabesiosis (BB) (Redwater, Tick Fever): Tick-borne disease of cattle.Caused by single-cell parasites mainly babesia bovis and babesiabigemina, with Rhipicephalus ticks being the major vector Rabies (RabiesVirus): Affects cattles and other ruminants It is transmitted throughthe biting of infected animals such as foxes, dogs, skunks and raccoons,but mostly by bat carrying rabies Neosporosis: Caused by the protozoanNeospora caninum. Affects cattle and sheep. Hosts are canids such asdogs and foxes Schmallenberg Virus (SBV): New emerging disease. Affectscattle, bison, sheep and goats. Transmitted through midges andvertically from dam to offspring Epizootic Hemorrhagic Disease Virus(EHDV): Most important infectious disease of white-tailed deer in US. Itaffects also antelope, mule and other deer species. Cattles are affecteduncommonly. It is spread by biting flies (midges, gnats) Lice: Affectscattle and other ruminants. Two types of lice, biting and sucking liceMange: Cattles and other ruminants are infected by mites Pseudocowpox:Caused by a parapox virus. Most common infectious cause of teat diseasein cattle Ringworm: Skin disease affecting cattles and other ruminantsIt is caused by Trichophyton verrucosum fungi Ulcerative mammillitis.Affects cattle. Caused by a herpes virus (BHV-2) Orf disease: Affectsprimarily sheep and goats. Caused by a parapox virus Toxoplasmosis:Affects sheep. Caused by the Toxoplasma gondii parasite. Coccidiosis:Affects cattle, sheep, chicken, dogs. Caused by Coccidian parasitesMyiasis: Parasitic infestation of a live mammal by fly larvae (maggots).Affects a wide range of mammals such as humans, sheep, horse, rabbitLouping ill: Acute, tick-transmitted viral disease that affects goats,horses, dogs, pigs, sheep, cattle. Caused by louping ill virusEchinococcosis: Affects sheep goats, cattle, swine, kangaroos, canidssuch as dogs and foxes, cats and wild felids. Parasitic disease causedby infection with tiny tapeworms of the genus EchinocococcusFasciolosis: Parasitic worm infection caused by the common liver flukeFasciola hepatica as well as by Fasciola gigantica. Affects human, sheepand cattle. It is a plant-borne zoonosis Coenurosis: Parasitic infectionthat develops in the intermediate hosts of some tapeworm species (Taeniamulticeps, T. serialis, T. brauni, or T. glomerata) and are caused bythe coenurus, the larval stage of these worms. Affects sheep and otherungulates but also humans Caprine arthritis and encephalitis Virus(CAEV): Affects goats Chagas (TRYPANOSOMA CRUZI): Affects human, horses,cattle and goats. Caused by the parasites trypanosomes Myxomatosis:Caused by Myxoma virus, transmitted through insect (mosquito, fly, furmite) bites. Affects rabbits Ear mites (canker): Affect rabbits. Causedby the mite Psoroptes cuniculi. Encephalitozoon Cuniculi: Affectrabbits. Caused by single-cell protozoan parasite Fleas: EctoparasitesRabbitpox: Affects rabbits. Caused by rabbitpox virus (RPXV) ViralHaemorrhagic Disease: Also known as rabbit Haemorrhagic Disease (RHD).Caused by a calicivirus. Affects rabbits Swine Influenza: Affects pigs.Cause by Swine Influenza virus (SIV) Japanese B Encephalitis Virus (JE):Affects pigs, transmitted through mosquitoes Trichinosis: Parasiticdisease caused by roundworms of Trichinella. Affects pigsEncephalomyocarditis Virus (EMCV): Affects pigs, transmitted throughrats Swine pox: Caused by Swine pox virus, affects pigs PorcineParvovirus Infection (PPV): Most common and important cause ofinfectious infertility in pigs Porcine Respiratory Corona VirusInfection (PRCV) Porcine Cytomegalovirus Infection (PCMV) TransmissibleGastro Enteritis (TGE): Caused by a coronavirus. Affects pigsEnteroviruses, SMEDI: gut-borne viruses. Affects pigs Aujeszky's disease(AD): Caused by a herpes virus, affects pigs Nipah virus disease: Causesdeath both in humans and pigs. New disease, first identified in Malaysiain 1998. Caused by a previously unknown pammyxovirus Swine Fevers;African, Classical, Hog Cholera Viruses: Affects pigs Teschen Disease:Caused by a porcine enterovirus serotype 1

TABLE 5 Cfu counting of Ag1 from shrimp intestines, weeks post-feedingtime postfeeding cfu/gm 0 4.8E+06 1 week 9.2E+06 2 weeks 4.5E+07 3 weeks4.2E+05 4 weeks 3.3E+05 5 weeks 6.4E+07 7 weeks 1.6E+06

Sequence Listings

As noted above, the instant application contains a full Sequence Listingwhich has been submitted electronically in ASCII format and is herebyincorporated by reference in its entirety. The following sequences arefurther provided herewith and are hereby incorporated into thespecification in their entirety:

DNA dsWsv477 Artificial SEQ ID NO. 1ATGTATATCTTCGTCGAAGGTTCCCCCCTCACAGGGAAGAGTTCATGGATGTCCAAGTTGATAGATACAGGATCATGTGGAATGTCTTTCCTCAATTTTCTTCGTATGAACACTTCTGACTACTACAACTGGCCTGCCGAAATCGGGACAGAACATCTCCAGTTAGGTTTCAGAGAAACCAGAGTGGTGGATGGAATGTTTGAACCTGTCCTAAAGACCTTTGTCGACTCGTGGAAGAAAGAGCAAGGAAAAGAGAGTTTGAAGGAATATCTGGACTACAACGGCCAAGTCATGGAGATCTACATCGCAGAATGGTTGAGACAAAGGCCACTAGCCTTCCACGTGTTTACCTATACAGATGAAGCTGTCAAGAGTGGATTCTTGAACGAGGAGGATCTAGATATGGATACTGCAACCAAGTGGATGGCTGAAATTATTAGAGAGAAGAGGGGCAATATTCAAGAAATAAAAGTGACCCCTAGAGTAGTCTTCAATGGCAATGGTTGTAGTGCATGTTTCTCTAACACTAAGAGAAACTTGTATAACTTTGGAACAAACTATAACAATGTTGTACATTGTGATTTGTTGTGCCCTTTTGCAAGGCATAGGATTGTACATTTCTTATAA DNA dsVp28Artificial SEQ ID NO. 2TCACTCTTTCGGTCGTGTCGGCCATCCTCGCCATCACTGCTGTGATTGCTGTATTTATTGTGATTTTTAGGTATCACAACACTGTGACCAAGACCATCGAAACCCACACAGGCAATATCGAGACAAACATGGATGAAAACCTCCGCATTCCTGTGACTGCTGAGGTTGGATCAGGCTACTTCAAGATGACTGATGTGTCCTTTGACAGCGACACCTTGGGCAAAATCAAGATCCGCAATGGAAAGTCTGATGCACAGATGAAGGAAGAAGATGCGGATCTTGTATCACTCCCGTGGAGGGCCGAGCACTCGAAGTGACTGTGGGGCGGAATCTCACCTTTGAGGGGACATTCAAGGTGTGGAACAACACATCAAGAAAGATCAACATCACTGGTATGCAGATGGTGCCAAAGATTAACCCATCAAAGGCCTTTGTCGGTAGCTCCAACACCTCCTCCTTCACCCCCGTCTCTATTGATGAGGATGAAGTTGGCACCTTTGTGTGTGGTACCACCTTTGGCGCACCAATTGCAGCTACCGCCGGTGGAAATCTTTTCGACATGTACGTGCACGTCACCTACTCTGGCACTGAGACCGAGTAA DNA dsVp19 Artificial SEQ ID NO. 3GCCACCACGACTAACACTCTTCCTTTCGGCAGGACCGGAGCCCAGGCCGCTGGCCCTTCTTACACCATGGAAGATCTTGAAGGCTCCATGTCTATGGCTCGCATGGGTCTCTTTTTGATCGTTGCTATCTCAATTGGTATCCTCGTCCTGGCCGTCATGAATGTATGGATGGGACCAAAGAAGGACAGCGATTCTGACACTGATAAGGACACCGATGATGATGACGACACTGCCAACGATAACGATGATGAGGACAAATATAAGAACAGGACCAGGGATATGATGCTTCTGGCTGGGTCCGCTCTTCTGTTCCTCGTTTCCGCCGCCACCGTTTTTATGTCTTACCCCAA DNA dsLUC Artificial SEQ ID NO. 4ACAGCCTGGGCATCAGCAAGCCCACCATCGTGTTCAGCAGCAAGAAGGGCCTGGACAAAGTCATCACCGTGCAGAAAACCGTGACCACCATCAAGACCATCGTGATCCTGGACAGCAAGGTGGACTACCGGGGCTACCAGTGCCTGGACACCTTCATCAAGCGGAACACCCCCCCTGGCTTCCAGGCCAGCAGCTTCAAGACCGTGGAGGTGGACCGGAAAGAACAGGTGGCCCTGATCATGAACAGCAGCGGCAGCACCGGCCTGCCCAAGGGCGTGCAGCTGACCCACGAGAACACCGTGACCCGGTTCAGCCACGCCAGGGACCCCATCTACGGCAACCAGGTGTCCCCCGGCACCGCCGTGCTGACCGTGGTGCCCTTCCACCACGGCTTCGGCATGTTCACCACCCTGGGCTACCTGATCTGCGGCTTCCGGGTGGTGATGCTGACCAAGTTCGACGAGGAAACCTTCCTGAAAACCCTGCAGGACTACAAGTGCACCTACGTGATTCTGGTGCCCACCCTGTTCGCCATCCTGAACAAGAGCGAGCTGCTGACA Amino Acid vp19White spot syndrome virus SEQ ID NO. 5MATTTNTLPFGRTGAQAAGPSYTMEDLEGSMSMARMGLFLIVAISIGILVLAVMNVWMGPKKDSDSDTDKDTDDDDDTANDNDDEDKYKNRTRDMMLLAGSALLFLVSAATVFMSYPKRRQ DNA vp19White spot syndrome virus SEQ ID NO. 6ATGGCCACCACGACTAACACTCTTCCTTTCGGCAGGACCGGAGCCCAGGCCGCTGGCCCTTCTTACACCATGGAAGATCTTGAAGGCTCCATGTCTATGGCTCGCATGGGTCTCTTTTTGATCGTTGCTATCTCAATTGGTATCCTCGTCCTGGCCGTCATGAATGTATGGATGGGACCAAAGAAGGACAGCGATTCTGACACTGATAAGGACACCGATGATGATGACGACACTGCCAACGATAACGATGATGAGGACAAATATAAGAACAGGACCAGGGATATGATGCTTCTGGCTGGGTCCGCTCTTCTGTTCCTCGTTTCCGCCGCCACCGTTTTTATGTCTTACCCCAAGAGGAGGCAGTAA Amino Acid vp28White spot syndrome virus SEQ ID NO. 7VTKTIETHTDNIETNMDENLRIPVTAEVGSGYFKMTDVSFDSDTLGKIKIRNGKSDAQMKEEDADLVITPVEGRALEVTVGQNLTFEGTFKVWNNTSRKINITGMQMVPKINPSKAFVGSSNTSSFTPVSIDEDEVGTFVCGTTFGAPIAATAGGNLFDMYVHVTYSGTETE DNA vp28White spot syndrome virus SEQ ID NO. 8GTGACCAAGACCATCGAAACCCACACAGACAATATCGAGACAAACATGGATGAAAACCTCCGCATTCCTGTGACTGCTGAGGTTGGATCAGGCTACTTCAAGATGACTGATGTGTCCTTTGACAGCGACACCTTGGGCAAAATCAAGATCCGCAATGGAAAGTCTGATGCACAGATGAAGGAAGAAGATGCGGATCTTGTCATCACTCCCGTGGAGGGCCGAGCACTCGAAGTGACTGTGGGGCAGAATCTCACCTTTGAGGGAACATTCAAGGTGTGGAACAACACATCAAGAAAGATCAACATCACTGGTATGCAGATGGTGCCAAAGATTAACCCATCAAAGGCCTTTGTCGGTAGCTCCAACACCTCCTCCTTCACCCCCGTCTCTATTGATGAGGATGAAGTTGGCACCTTTGTGTGTGGTACCACCTTTGGCGCACCAATTGCAGCTACCGCCGGTGGAAATCTTTTCGACATGTACGTGCACGTCACCTACTCTGGCACTGAGAC CGAGAmino Acid Wsv477 White spot syndrome virus SEQ ID NO. 9MYIFVEGSPLTGKSSWMSKLIDTGSCGMSFLNFLRMNTSDYYNWPAEIGTEHLQLGFRETRVVDGMFEPVLKTFVDSWKKEQGKESLKEYLDYNGQVMEIYIAEWLRQRPLAFHVFTYTDEAVKSGFLNEEDLDMDTATKWMAEIIREKRGNIQEIKVTPRVVFNGNGCSACFSNTKRNLYNFGTNYNNVVHCDLLCPFARHRIVHFL DNA Wsv477 White spot syndrome virus SEQ ID NO. 10ATGTATATCTTCGTCGAAGGTTCCCCCCTCACAGGGAAGAGTTCATGGATGTCCAAGTTGATAGATACAGGATCATGTGGAATGTCTTTCCTCAATTTTCTTCGTATGAACACTTCTGACTACTACAACTGGCCTGCCGAAATCGGGACAGAACATCTCCAGTTAGGTTTCAGAGAAACCAGAGTGGTGGATGGAATGTTTGAACCTGTCCTAAAGACCTTTGTCGACTCGTGGAAGAAAGAGCAAGGAAAAGAGAGTTTGAAGGAATATCTGGACTACAACGGCCAAGTCATGGAGATCTACATCGCAGAATGGTTGAGACAAAGGCCACTAGCCTTCCACGTGTTTACCTATACAGATGAAGCTGTCAAGAGTGGATTCTTGAACGAGGAGGATCTAGATATGGATACTGCAACCAAGTGGATGGCTGAAATTATTAGAGAGAAGAGGGGCAATATTCAAGAAATAAAAGTGACCCCTAGAGTAGTCTTCAATGGCAATGGTTGTAGTGCATGTTTCTCTAACACTAAGAGAAACTTGTATAACTTTGGAACAAACTATAACAATGTTGTACATTGTGATTTGTTGTGCCCTTTTGCAAGGCATAGGATTGTACATTTCTTATAA

What is claimed is: 1-7. (canceled)
 8. A genetically modified probioticbacteria configured for the biocontrol of white spot syndrome virus inshrimp comprising: a genetically modified probiotic enteric bacteriathat expresses at least one heterologous inhibitory polynucleotideselected from the group consisting of: SEQ ID NO. 1, SEQ ID NO. 2,and/or SEQ ID NO. 3, wherein said at least one heterologous inhibitorypolynucleotide is double stranded RNA polynucleotide molecule (dsRNA)that targets an essential gene in a white spot syndrome virus, andwherein said genetically modified probiotic enteric bacteria isconfigured to be probiotic to shrimp.
 9. A genetically modifiedprobiotic bacteria configured for the biocontrol of white spot syndromevirus in shrimp as described in claim 8 wherein said geneticallymodified probiotic enteric bacteria comprises a genetically modifiedBacillus subtilis bacteria.
 10. A genetically modified probioticbacteria configured for the biocontrol of white spot syndrome virus inshrimp as described in claim 9 wherein said genetically modifiedBacillus subtilis bacteria comprises an RNaseIII deficient geneticallymodified Bacillus subtilis bacteria.
 11. A genetically modifiedprobiotic bacteria configured for the biocontrol of white spot syndromevirus in shrimp as described in claim 10 wherein said essential gene ina white spot syndrome virus comprises an essential gene in a white spotsyndrome virus selected from the group consisting of: the vp28 gene, thevp19 gene, or the wsv477 gene.
 12. A genetically modified probioticbacteria configured for the biocontrol of white spot syndrome virus inshrimp as described in claim 11 wherein said at least one heterologousinhibitory polynucleotide selected from the group consisting of SEQ IDNO. 1, SEQ ID NO. 2, and/or SEQ ID NO. 3, comprises at least oneheterologous inhibitory polynucleotide selected from the groupconsisting of: at least one heterologous inhibitory polynucleotidehaving at least 75% homology with SEQ ID NO. 1; at least oneheterologous inhibitory polynucleotide having at least 75% homology withSEQ ID NO. 2, and/or at least one heterologous inhibitory polynucleotidehaving at least 75% homology with SEQ ID NO.
 3. 13. A geneticallymodified probiotic bacteria configured for the biocontrol of white spotsyndrome virus in shrimp as described in claim 12 wherein said shrimpcomprises shrimp raised in an aquaculture environment.
 14. A geneticallymodified probiotic bacteria configured for the biocontrol of white spotsyndrome virus in shrimp as described in claim 13 wherein saidgenetically modified probiotic enteric bacteria that expresses at leastone heterologous inhibitory polynucleotide selected from the groupconsisting of SEQ ID NO. 1, SEQ ID NO. 2, and/or SEQ ID NO. 3 istransmitted to a shrimp progeny through vertical transmission.
 15. Agenetically modified probiotic bacteria configured for the biocontrol ofwhite spot syndrome virus in shrimp as described in claim 14 and furthercomprising the step: administering, in vivo, a therapeutically effectiveamount of said genetically modified probiotic bacteria to said shrimpraised in an aquaculture environment such that said heterologousinhibitory polynucleotide configured to downregulate expression of atleast one essential pathogen gene induces an RNA interference mechanismcausing the downregulation of said essential gene in a white spotsyndrome virus and results in a reduction in mortality due to WSSV insaid shrimp raised in an aquaculture population by approximately between1% and 50% when administered to shrimp in an aquaculture environment ina therapeutically effective amount.
 16. (canceled)
 17. A geneticallymodified probiotic bacteria configured for the biocontrol of white spotsyndrome virus in shrimp comprising: a genetically modified RNaseIIIdeficient probiotic bacteria that expresses at least one heterologousinhibitory polynucleotide selected from the group consisting of SEQ IDNO. 2, wherein said at least one heterologous inhibitory polynucleotideis double stranded RNA polynucleotide molecule (dsRNA) that targets thevp28 gene in a white spot syndrome virus identified as SEQ ID NO. 8, andwherein administration of a therapeutically effective amount of saidgenetically modified RNaseIII deficient probiotic bacteria to a shrimpresult in a reduction in mortality due to white spot syndrome virus. 18.(canceled)
 19. An aquatic organism ingestible compound for thebiocontrol of white spot syndrome comprising: a treated feed for aquaticorganisms having a genetically modified probiotic bacteria thatexpresses at least one heterologous inhibitory polynucleotide selectedfrom the group consisting of: SEQ ID NO. 1, SEQ ID NO. 2, and/or SEQ IDNO. 3, wherein said at least one heterologous inhibitory polynucleotideis double stranded RNA polynucleotide molecule (dsRNA) that targets anessential gene in a white spot syndrome virus.
 20. An aquatic organismingestible compound for the biocontrol of white spot syndrome asdescribed in claim 19 wherein said genetically modified probioticbacteria comprises a genetically modified probiotic Bacillus subtilisbacteria.
 21. An aquatic organism ingestible compound for the biocontrolof white spot syndrome as described in claim 20 wherein said geneticallymodified probiotic Bacillus subtilis bacteria comprises an RNaseIIIdeficient genetically modified probiotic Bacillus subtilis bacteria. 22.An aquatic organism ingestible compound for the biocontrol of white spotsyndrome as described in claim 21 wherein said essential gene in a whitespot syndrome virus comprises an essential gene in a white spot syndromevirus selected from the group consisting of: the vp28 gene, the vp19gene, or the wsv477 gene.
 23. An aquatic organism ingestible compoundfor the biocontrol of white spot syndrome as described in claim 22wherein said at least one heterologous inhibitory polynucleotideselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, and/orSEQ ID NO. 3, comprises at least one heterologous inhibitorypolynucleotide selected from the group consisting of: at least oneheterologous inhibitory polynucleotide having at least 75% homology withSEQ ID NO. 1; at least one heterologous inhibitory polynucleotide havingat least 75% homology with SEQ ID NO. 2, and/or at least oneheterologous inhibitory polynucleotide having at least 75% homology withSEQ ID NO.
 3. 24. An aquatic organism ingestible compound for thebiocontrol of white spot syndrome as described in claim 23 wherein saidaquatic organisms comprises shrimp.
 25. An aquatic organism ingestiblecompound for the biocontrol of white spot syndrome as described in claim24 wherein said shrimp comprise shrimp raised in an aquacultureenvironment.
 26. An aquatic organism ingestible compound for thebiocontrol of white spot syndrome as described in claim 25 wherein saidgenetically modified probiotic bacteria that expresses at least oneheterologous inhibitory polynucleotide selected from the groupconsisting of SEQ ID NO. 1, SEQ ID NO. 2, and/or SEQ ID NO. 3, istransmitted to a shrimp progeny through vertical transmission.
 27. Anaquatic organism ingestible compound for the biocontrol of white spotsyndrome as described in claim 26 and further comprising the step:administering, in vivo, a therapeutically effective amount of saidgenetically modified probiotic bacteria to said shrimp raised in anaquaculture environment such that said heterologous inhibitorypolynucleotide configured to downregulate expression of at least oneessential pathogen gene induces an RNA interference mechanism causingthe downregulation of said essential gene in a white spot syndrome virusand further results in a reduction in mortality due to WSSV in saidshrimp raised in an aquaculture population by approximately between 1%and 50% that are administered a therapeutically effective amount of saidtreated feed
 28. A shrimp ingestible compound for the biocontrol ofwhite spot syndrome comprising: a treated feed for shrimp having agenetically modified probiotic enteric bacteria that expresses at leastone heterologous inhibitory polynucleotide selected from the groupconsisting of SEQ ID NO. 1, SEQ ID NO. 2, and/or SEQ ID NO. 3, whereinsaid at least one heterologous inhibitory polynucleotide is doublestranded RNA polynucleotide molecule (dsRNA) that targets an essentialgene in a white spot syndrome virus.
 29. A shrimp ingestible compoundfor the biocontrol of white spot syndrome as described in claim 28wherein said genetically modified probiotic enteric bacteria comprises agenetically modified probiotic Bacillus subtilis bacteria.
 30. A shrimpingestible compound for the biocontrol of white spot syndrome asdescribed in claim 29 wherein said Bacillus subtilis bacteria comprisesan RNaselll deficient genetically modified probiotic Bacillus subtilisbacteria.
 31. A shrimp ingestible compound for the biocontrol of whitespot syndrome as described in claim 30 wherein said essential gene in awhite spot syndrome virus comprises an essential gene in a white spotsyndrome virus selected from the group consisting of: the vp28 gene, thevp19 gene, or the wsv477 gene.
 32. A shrimp ingestible compound for thebiocontrol of white spot syndrome as described in claim 31 wherein saidat least one heterologous inhibitory polynucleotide selected from thegroup consisting of SEQ ID NO. 1, SEQ ID NO. 2, and/or SEQ ID NO. 3comprises at least one heterologous inhibitory polynucleotide selectedfrom the group consisting of: at least one heterologous inhibitorypolynucleotide having at least 75% homology with SEQ ID NO. 1; at leastone heterologous inhibitory polynucleotide having at least 75% homologywith SEQ ID NO. 2, and/or at least one heterologous inhibitorypolynucleotide having at least 75% homology with SEQ ID NO.
 3. 33. Ashrimp ingestible compound for the biocontrol of white spot syndrome asdescribed in claim 32 wherein said shrimp comprise shrimp raised in anaquaculture environment.
 34. A shrimp ingestible compound for thebiocontrol of white spot syndrome as described in claim 33 wherein saidgenetically modified probiotic enteric bacteria that expresses at leastone heterologous inhibitory polynucleotide selected from the groupconsisting of SEQ ID NO. 1, SEQ ID NO. 2, and/or SEQ ID NO. 3 istransmitted to said shrimp progeny through vertical transmission.
 35. Ashrimp ingestible compound for the biocontrol of white spot syndrome asdescribed in claim 34 and further comprising the step of administering,in vivo, a therapeutically effective amount of said genetically modifiedprobiotic bacteria to said shrimp raised in an aquaculture environmentsuch that said heterologous inhibitory polynucleotide configured todownregulate expression of at least one essential pathogen gene inducesan RNA interference mechanism causing the downregulation of saidessential gene in a white spot syndrome virus an further results in areduction in mortality due to WSSV in said shrimp raised in anaquaculture population by approximately between 1% and 50%. 36-73.(canceled)